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 17.8.1913-6.2.199B

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 SEVENTH EDITION

c 4 n a n tb a n a rg y a n
,

ancJ ^ ^ n i k e r ' s

Textbook
of
Microbiology

T h l t uno

lfq t - 1 -E 09
jU iy righted
C o p y rig h te d m aterial
 m o n th a n o r a y o n
and
a n i k e r 's

Textbook
of
Microbiology

(Late) ft Ananthanoroyom

CK Joyarom Paniker

CK Joyoram Poniker

^ 7
Orient Longman

C o p y rig h te d m aterial
p#
Trinity College London
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C o p y rig h te d m aterial
Preface to the Seventh Edition

Only four yrai5 have puud since the si vth e-hdon tfTcxtbookofMkrt ewas publ rhod, bur rapid developments
in [lie subject have made a new edition necessary. During this short period, new infectious disease!; have emerged
f>r rc-emcrged in new forms. For w-i tuple, Severe Acute Respiratory Syndrome (SARS) vims appeared suddenly
cau sing death and panic in many countries, and the bin I FIll virus posed repeated pandemic threats.

Microbiology has become an increasingly important discipline, set tn face new challenges- Exciting .iih .m. ■: > in
diagnostic microbiology using sophisticated techniques can help in the rapid identification offLCW pathogens and
serve to contain them. TTu- was shown by the idemfLcaiiunofthe new S A R S virus within weeks by concerted multfr
disciplinary international efforts. W : 11 II; such ■,. ienrifie progress is A boon, some of it can be potentially dangerous, as
lor instance in the recent case of chemical synthesis of a complete pathogenic pnlioviru-. in the laboratory.

In rlii* edition, the seventh, relevant new information has been added and all chapters revised and updated,
nia mmiiii.n.g the general format of the book, rfhe "IcrtfjooJt ttfMicr^iofogy ha -been in use now for more than a
quarter of a . eutury It has benefited gjeady from the comments and suggestions from students, teachers and other
readers.
Tlicir help is gratefully acknowledged.

C.K. Jayafarn Paniker

ShiinM, 1/3833L E^r Hit] K^d.
L’dk-ur. Kerala 617 006.

Preface to the First Edition

Many of the health problems in developing countries like Tndis art diflfercm from those of developed countries.
Hi. i■■:;iI diseases still play a considerable role in diseases in our country. J'opics such as cholera and enteric diseases
are important to us though only ofless or academic interest to the developed countries. The Increasing importance
of the newer knowledge in immunology to health and disease is not adequately stressed in most of the extant
texthonks. Virus-diseases whi, h are responsible for nearly bOper cent of human Illness requite wider coverage, The
general approach to the teaching (dmicrobiology intiur country has also been rather static. All these factors .ailed for
a Tcxrhonk of mii^oblolngy more ■■.illI co ctHim11vs like India

We therefore undertook rhis endeavour based on our experience of teaching Urnki^radlJiHes and postgriduites for
over two decades. Wt omitted the discipline of parasitology from our book since we jiLcady have an excellent
textbook on the subject published in India.

Th is book has taken us over throe years to wr ire and over a year in publicat i:>n. N aturally we would be out of date to
a certain and inevitable extent. Vfe do not cbnm any perfection On the contrary we have nequesred medical
Students and teachers ill over the country Du wiile to us about any shortcomings and give us suggesti ons as to how
tD improve the book. We shall spare no pain- in. seeing that their valuable suggestions arc ^nvi: effect to ifi OUf
■'uvortdwii rf.ijci,

R. Ananthanarayan
C.K. Jayaram Paniker

C o p y rig h te d m aterial
 Acknowledgements
For kind permi5-sion to use illustrations, supply of photographs, helpful suggestion* Arid advice, the author is
indebted to the following organisations and persons:
Wirld Health Ot^aniiarion; Indian Cnuncil of Medical R sw ch l National Institute of Vinuto^ Punci Dr. G.
Balkrith Nsur. Du A_N. Ghosh. Dr. T N . Naik and D r Trivertl Krishr.an of National Institute of Cholera and
Enteric diseases, Calcutta. Prof, M, Muhui and Piot. T. Jacob John i>t Christian Medical College, WUore. Prof,
Anin Chi tale. Jaslolt Hospital, Mumbai; Dr. SJM.Sirsat, Can«r Research Institute. Mumbai: Piof M.D.Mathur,
Maulana Azad Medical College. New Delhi. Prof. J. Shanmugham, SCT Medical Centre, Trivandrum;
Dr. t ]iri;a G- Ran. Medical College, Calicut; Df. G.M. Warfce, Hi Media Laboratories, Mumbai.

C o p y rig h te d m aterial
 Contents

2W I
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C o p y rig h te d m aterial
26. Coryuebaeterium 231

1
I

51. Poxviruses 468

58. Rhabdovirusts 535

C o p y rig h te d m aterial
 :
61, Oncogenic Viruses 574
62, Human Immunodeficiency Virus: AIDS 582

/jfljS . ^fel^rrlM .J:'-' tjfa J iir .

64, Bacteriology of Water, Milt and Air 603
65, Medical Mycology 610
~jiWafnr} Con* d .-4

68, Hospital Infecrion 634

C o p y rig h te d m aterial
C o p y rig h te d m aterial
 Historical Introduction
Metti-CSl Riiaobinli]^ LHthe study of microbes that
infect human*, the diseases they causef their
diagnosis, prevenrion and treatment, lr also deals
with the response of the human hosr to microbial
and other antigens.
Disease and death have always held the
attention ol the human mind. Ancient humans
ssi- ri bed them to tliri ne wrath and Other supernatural
forces. Later, other concept!; such as the effect of
the environment, of bodily constitution and of faulty
diet were proposed. There have been, from very
earlvtimes, occasional suggestions that diseases may
result from invasion of the body by external
contagion. Varo and Columella in the first century
be ]HUlulated that diseases Were caused by invisible
beings {Animalia initmta), inhaled or ingested.
Fracaatotius of Verona (1546) proposed a
conngium vivum as a possible cause of infectious
disease and von P[em:L7. (1 767) suggested that each
disease was caused by a separate agent. Kirclur
(165£) reported finding minute worms in the blood
of plague victims, but with the equipment available
to him it is mono likely that what he observed were
only blood cells.
As microbes are invisible to rhe unaided eye,
definitive knowledge about them had to await the
development of microscopes, 'l'he credit for having
first observed and reported bacteria belongs to
Antony van Leeuwenhoek, a draper in Drift,
Holland, whose bobby was grinding lenses and
observing diverse materials Through them. In 1693
he made accurate descriptions of various types of
bacteria and communicated them ro the Royal
Society' of London. The significance ol these
observations was nnt realised then and to
Leeuwenhoek the world of ‘litlie tmiudftiJes’ afi
he called Therm, represented only a curiosity of
nature. ][ was only some two centuries later that
their importance in medicine and biology as a whole
came to he recognised.
The earliest discovery of a pathogenic
microorganism was probably made by Augustino
Bassi (183.5), who showed that the muscardtne
disease of silkworms was caused hv a fungus.
Davaine and Pullender (1850) observed anthrax
bacilli in the blood of animals dying of the disease.
In fact, even before the microbial etiology of
infections had been established, Oliver Wendell
Holmes in the t!SA (1S4.1) ami Ignaa SemmeDweis
in Vienna (1846) had independently concluded that
puerperal sepsis was contagious. Semmelwcis also
identified its mode of transmission hv doctors and
medical students attending on women in labour in
the hospital and had prevented it by rhe simple
measure of washing hands in ait antiseptic solution,
for which service to medicine and humanitvr he
was persecuted by medical orthodoxy and driven
insane.
The development of niterminology as a scientific
discipline dates from Louis Pasteur (1S22-95).
Though trained as a chemist, his stud io on
feiinecitation led him ro take an interest in
uncmogauisiTLS. He e>Lablished that fermentation
was the result of microbial activity and that different
types of fermentations were associated with different
types of microoganisms (1557). "l’hebasic principles
and Techniques of microbiology were evolved by
Pasteur during bis enquiry into the origin □f
C o p y rig h te d m aterial
2 * Texibou* a1 Microbiology *
microbes, This was then the subject of much
oontnoveny. Needham, an Irish priest, had lu 1745
published experiments purporting the spontaneous
generation (abiogene^i-s) of microorganisms in
pLitrescible fluids, This view was opposed by
Spallanzani, an Italian abbot (1769). In a series of
classic experiments, Pasteur proved conclusively that
all forms of life „ even microbes, arose only from
their like and not Je novo. In the course of these
studies, he introduced techniques of sterilisation
and developed the steam steriliser, hot ,iir oven and
autoclave. He also established the differing growth
needs of different bacteria. 1 lis work attracted such
attention and he attained such eminence in the
world of science that not only France hut all Europe
looked to him to r-ulve major problems in various
fields. Thus starred Ins studies on pebrine, anthrax,
chicken cholera and hydrophobia. An accidental
observation that chicken cholera bacillus culture*
left on the bench for several weeks lost their
pathogenic property but rttinned their abiluy to
protect the birds against subsequent infection by
them, led to the discovery of the process of
attenuation and the development of live vaccines.
He attenuated cultures of the anthrax bacillus by
incubation at high temperature (42-43 °C) and
proved that inoculation of such cultures in animals
induced specific protection against anthrax. I he
success of such immunisation was dramatically
demonstrated by a public experiment on a farm at
Pbuilty-le-Fort (18B1) during which vaccinated
sheep, goats and cows were challenged with a
virulent anthrax bacillus culture. All the vaccinated
animals survived the challenge, while in lc|UlhI
number ofunvaccinatedcontrol animal; succumbed
to it, it was Pasteur who coined the term mccrne
for such prophylacdc preparations to commemorate
the first of such preparations, namely cowpox,
employed by Jenner for protect ion against smallpox.
The greatest impact in medicine was made by
Pasteur's development of a vaccine for hydrophobia.
This was acclaimed throughout the world. The
Pasteur Institute, Pans was built by public
Contributions and similar institutions were
established soon in many other countries for the
preparation of vaccines and for the investigation of
infectious diseases
An nmncd iate appl icat o n of Pasteur's work was
the introduction of am isepuc techniques in surgery
by bister { I H67) effecting a pronounced drop in
mortality and morbidiry due to surgical sepsi-.
Lister's antiseptic surgery involving the use of
carbolic acid was cumbersome and ha^.Lrdous but
was a milestone in the evolution nfsurgical pradke
from the era of 'laudable pus’ to modern aseptic
techniques.
While Pasteur in France laid the foundations
of microbiology, Robert Koch (1 8 4 3 -1 9 1 0 ) in
Germany perfected bacteriological techniques
during his studies on the culture and life cycle of
the anthrax bacillus (1976). He introduced staining
techniques and methods of obtaining bacteria ill
pure culture using solid media. He discovered the
bacillus of tuberculosis (1882) and the cholera
vibrio (1883),
Pasteur and Koch attracted many girted disciples
who discovered the causative agents of several
bacterial infection!; Ind enlarged the scope and
content of microbiology by their labours. In 1874
Hansen described the leprosy bacillus; in 1979
Neisaer described the gonococcus; in 1881 Ogston
discovered the staphylococcus; in 1884 LocHlcr
isolated the diphtheria bacillus; in 1984 Nicolaicr
observed the tetanus bacillus in soil; in 1886
Fracnkel described the pneumococcus; in 1887
Biucc identified the causative agent of Malta fever,
m 1905 Schaudlnn and Hoffmann discovered the
spirochete d) syphilis.
R|)1 I\ and Vers in (1883) identified a new
mechanism of pathogenesis when they discovered
the diphtheria toxin. Similar toxins were identified
in tetanus and some other bacceria. The toxins were
found to be specifically neutralised by their
anatoxins, Ehrlich who studied toxins and antitoxins
in quantitative terms laid the foundations of
biological standardisation.
C o p y rig h te d m aterial
 n Historical liltrufluc^nn ►
The causative agents of various infectious
di-icascs were being reported by different
investigators '.n such profit-ion tha: i.t was necessary
to introduce criteria for proving the claims that a
niLcir ion'.mi-nil i isolated Itom a disease was indeed
causal)v related to it. Thene criteria, first indicated
by Henle, were enunciated by Koch and arc known
as Koch's postulates. According to these, a
microorganism can be accepted as the causative
agenr of an infectious disease only if the following
ainJil ions afe satisfied:
1. The bacterium should be constantly associated
with the lesions of the disease.
2. It should be possible tu isolate the bacterium in
pure culture from the lesions.
3. Inoculation of such pure culture into suitable
laboratory animals should reproduce the lesions
of the disease.
4. It should be possible to neisolate the bacterium
in pure culture from the lesions produced in
the experimental animals.
An additional criterion introduced subsequently
requires that specific antibodies to the bacterium
should be demonstrable in the scrum of patients
suffering from the disease. Though it may not
always he possible to satisfy all the postulates in
every ease, they have proved extremely useful in
fitting doubtful claims made regarding the causative
agents of infectious diseases.
By the begi nni.ng of the twentieth century, many
infectious di-cases had been proved to be caused
by bacteria. Bur there remained a large number of
disease?; such as smallpox, chickertpOx, measles,
■n lliirm and the common cold for which no
ruutcri.il cause could he established. During ins
lnvesiiglliuit of rabies in d o ^ hPasteur had suspected
that the disease could be -caused hy a microbe too
small to be seen even under the microscope. The
existence of such ultram croscopic microbes was
proved when Jvanuvslw ( Lfi^l reproduced mosaic
disease in the tobacco plant, by applying to healthy
leaves juice from the diseased plants from which
all bacteria had been removed by passage through
3
fine filters, Beijerinck (1898) confirmed these
findings and coined the term v.'-rns for nuch filterable
infectious agents. Locfflcr and Frosch (1898)
observed rhat the foot and mouth disease of cattle
was caused hy a similar filter-passing virus. The
first human di-case proved to have a viral el i-.ilo^v
was yellow fever, I ’he US Army Commission under
Walter Reed, invesrigating yellow fever in Cuba
i.l902) established not only that it was caused hy a
filterable virus but also rhat ir was transmitted
through the ii ite of 11ifected rflO»|uitoes. 1-indtfc 11«r
and Popper (1909) showed that poliomyelitis was
caused bv a filterable virus and transmitted the
di-ease experimentally to monkeys. Investigjirinn of
viruses and the diseases caused by them was
rendered difficult as viruses could not be vi-iualised
under the light microscope or grown in culture
media. Though the larger viruses coy id be seen
after appropriate staining under the light
microscope, detailed study of their morphology hod
to wait till the introduction of the electron
microscope by Ruska (1 9 3 4 } and subsequent
refinements in electron microscopic techniques.
Cultivation of viruses was possible onl} in n f naals
or in human volunteers rill the technique of
growing them on chick embryos was developed by
Goodpasture in the 1930s, The application of tissue
culture in virology expanded the scope of virologies!
technique-, con-,iderablv.
The possibility' that virus infection could lead
to malignancy was first put forth hy Kllcrman and
Bang (1908), Peyton Rous (1911) isolated a virus
causing sarcoma in fowls. Several viruses have since
been isedated which cause natural and experimental
Honour*, in animals and birds. Viruses also cause
rratiign&nt transformation ud infected cells in tissue
culture. The discovery of viral and cellular
oncogenes has shed light on the possible
mechanisms of viral oncogenesis. After many
decades of futile search, positive proof of a virus
causing human malignancy was established when
the virus of human I -ceil leukemia was isolated in
1980,
C o p y rig h te d m aterial
4 « Textbook v f/tcrobiatogy *
Twmrt (1 9 1 5 ) and d'Hercllc (1917)
independently discovered. a Iv'i-L phenomenon in
bacterial cultLirCJ. The age ills Responsible Wetc
termed bacteriophage* - viruses that attack bacteria.
Early hopes that bacteriophages may have
therapeutic appl Virions had to be abandoned but
these viruses have paid, unexpected, scientific
dividends. The essential part of viruses is their core
of nucleic acid which acts as the Cartier of generic
information in the same manner as in higher
organisms. The discipline of molecular biology owes
its origin largely to studies on the genetics ol
bacteriophages and bacteria.
It had been noticed from very early days [hat
persons surviving an attack of smallpox did not
develop the disease when exposed to the infection
subsequently. This observation had been applied
lor the prevention of the disease by producing a
mild form of smallpox intend orally (variolation).
Tb.-.-i practice, prevalent in India, China and other
am lent civilisation* from time immemorial, was
introduced in England by Lady Mary Worthy
Montague (1718) who had observed the custom
in Turkey. Variolation was effective but hazardous,
lenner, observing the immunity to smallpox in
milkmaids who were exposed to occupational
cowpox infection, introduced the technique of
vaccina I .on living CoWpuX material (l796).Thi-. was
the first distance of scientific immunisation and,
though introduced empirically, has stood the test
of Time. Jenner’s vaccination paved the way for the
ultimate eradication of smallpox.
The next major discovery in iinmuriry was
Pasteur's development of vacuncs for chicken
cholera, anthrax and rabies. While the techniques
introduced by him were successful, the mechanism
of protection afforded by them remained obscure.
The cxplanac ion of the underlying mechanism came
from two sources. Nuttall (13SS) observed that
defibrinated blood had a bacteikidal effect, and
Buchner (13 8 9 ) noticed that this effect was
abolished by heating the sera, for one hour at 55 °C.
The heat labile bactericidal factor was termed
'aJcxinc', A specific humoral factor or 'anriborfy'
was described by von Belu mg and Kitasaio (1890)
in the serum of animals which had received
sublethal doses of tetanus [oxin. Pfeiffer (1893)
demonstrated hactcri filial effect in vivo by injecting
live chokra vibrios irtrapcntoncally in guinea pigs
previously Injected with killed vibrios. The vibrios
were shown to undergo lysis. The humoral nature
of such lytic activity was proved by Bordet (18951,
who defined the two components part ic ipa Ting in
the reaction, the first being heat stable and found
in immune sera (an Libody or xuba ta nuc
scnsr&jTicafricc) and the second being heat labile
and identical with Buchner's aJexine, subsequently
named 'aimyifcmen f . Soon a number nfother ways
were demonstrated in which antibodies react with
antigens, such as agglutination, precipitation,
complement fixation and neutralisation,
Metchnikoff (1$83) discovered the phenomenon
of phagocytosis and proposed the phagocytic
response as the pn me defence ag-i inst the ti icrob i4
invasion of tissues. This led to the cellular concept
of immunity. Polemics regarding the significance
of the cellular and humoral mechanisms of
immunity were hugely put to rest with the discovery
by W right (1903) of opsonization, in which
antibodies and phagocytic cells act :.n conjunction.
Prior experience with a microorganism or other
antigen did not always result in the beneficial eflect
of immunity or protection. At rimes it caused the
opposite effect. EGoch (1B90) had noticed that when
1 11 l1tuhercle hacilhis or it- protein was injected into
a guinea pig already infected with the bacillus, an
exaggerated response took place - a hypersensitivity
reaction known as Koch’s phenomenon. Puttier
and Richer (1902), studying the effect of the tone
extracts of sea anemones in dogs made the
paradoxical observation that dogs which had prior
contact with the [amn were abnormally sensitive
to even minute quantities of it subsequently. This
phenomenon was termed Later, many
similar reactions were observed, both
rape i imentally and in nature, of injury, disease or
C o p y rig h te d m aterial
 * historical Introduction *
even death resuming from repeated contacts wirh
antigens. T!w importance of this phenomenon in
the pathogenesis of many human diseases, led to
the development o f the discipline of allergy.
The characteristic feature of immunity,, whether
if is protective or destructive (as in allergy), is its
specificity. As the mediators of humoral immunity
(antiI>odies) are globulins, the explanation for the
ex;|ui>itL specificity ol the immunological reaction
had to awa ir the advances i n protein chcm is try. The
p^mccri ng work o f! ,andsteincr laid the foundations
of immunodiemistry. Chemists doi:mrated the study
of immunity for several decades, and theories of
antibody synthesis were postulated by them, which
sometlines ran counter To biological laws. In 1955,
lerne proposed the natural selection theory of
antibody synthesis which attempted to explain the
chcm it al specificity and biological basis of antibody
synthesis, signifying a return to the original views
of antil»ody formation proposed by Ehrlich (1S98).
Burnet (1957) modified this into the clonal
selection theory, a. concept which, with m inor
alterations, holds sway even now. The last few
decades have witnessed an explosion of conceptual
and technical advances in immunology.
Immunological processes in health and disease are
now better understood followi ng the identification
of the two components of immunity - the humoral
or antibody mediated processes and the cellular or
cell mediated processes — which develop and are
manifest in separate pathways.
Til] recently, a teleological view of immuniry
prevailed. It was considered a protective mechanism
designed to defend the body against invasion by
microorganisms. Based on die original suggestion
□f Thomas (1959), Burnet (1967) developed the
concept of JmniunoJngiVai ?urveillqncer according
to which the primary function of the immune
system is to preserve the integrity of the body,
seeking and destroying all ‘foreign1 antigens,
whether autogenous or external in origin.
Malignancy was visualised as a failure of this
function, and the scope of immunity was enlarged
5
to include natural defence against cancer. Another
aspect of this role of immunity is in the rejection of
homografft. Understanding of the immunological
basis o f transplantation, largely due to the work o f
Medawar and Burnet, made successful transplants
possible by elective immunosuppression and proper
selection of donors based on histocompatibility. The
history of transplantation thus runs parallel to the
history (if b lo o d transfusion, which was
unsuccessful and even fatal before the discovery of
blood groups by Landsteincr (1900).
In the early twem Lentil century, attempts were
made to exploit the immunological information
available by the development o f vaccines and sera
for the prophylaxis and Treatment o f infectious
diseases, 'fill Domagk (1935) initiated scientific
chemotherapy with the discovery of prontosil,
antisera were the only specific therapeutic agents
available for the management of infectious diseases.
Fleming (1929) made the accidental discovery that
the fungus Fcnidllium produces a substance which
destroys staphylococci. \%jrk no this at Oxford in'
Florey, Chain and their team during the Second W>rld
War led to the isolation of the active substance
penicdUlLn and its subsequent mass production. This
was the begi nni ng of the antibiotic cm- Other si i:i ilar
antibiotics were discovered in rapid mccessinri. With
the sudden availability of a wide range of antibiotics
with potent antibacterial activity, it was hoped that
bacfcn-.il infections would be controlled within a short
peri id. But so:hi the development of dm^ resistance
ni bacteria presented serious difficulties.
With the development of a wide variety of
antibiotics active against the whole spectrum of
path >genic bacteria, and of effective vaccines against
most viral diseases, expectations were raised about
the eventual elimination of all infectious diseases.
The global eradication of smallpox inspired visions
of similar campaigns against other major pestilences.
However, when new infectious diseases began to
appear, caused hv hitherto unknown micro­
organisms, or by known microbes producing novel
manifestations, it was realised that controlling
C o p y rig h te d m aterial
6 < Te>:lt>CGk Micros DlOfjy ►

microbes was a far more difficult task than was 1901 Emil A Von Behnng
imagined. The climax came in 1981 when AIDS 1902 Ronald Ross
was identified in the USA and began its pandemic 1905 Robert Koch
1907 C IA Lavieran
spread. Unceasing viy;i3 appears essential to protect
1906 Paul Ehrlich and Elie Metchnikoff
humans from microbes. 1913 Charles Richct
Apart bom the obvious benefits such as specific 1919 JuJes Bordet
methods of diagnosis, prevention and control of 1926 Johannes Fibiger
infectious diseases, medical microbiology has 1926 Charles NicoUc
1930 KaH Landsteiner
contributed to scientific knowledge and human
1939 Gerhardt Domagk
welfare in many other ways. Microorganisms
1945 Alexander Fleming, Howard Florey and EB
constitute the smallest forms of living brings and, Chain
therefore, have been employed as models of studies 1951 MaxTheiler
on genetics and huochemistty. As nature's laws are 1952 Sedman A Waksman
universal in application, informarinn derived from 1954 JF Euders, FC Robbins and TH WcLcr
1958 G W Beadle, Joshua Lederbeqr and EL Tatum
the investigation of miLTobcs holds true, i it the main,
1960 Macfarlane Burnet and Exeter Brian Mcdawar
for humans as well. 1965 Francois Jacobs Andre Lwoff and Jacques
Studies on microorganisms have contributed, Monod
more than anything else, to unravel!ing the genetic 1966 Peyton Rous
code and other mysteries of biology at the molecular 1969 M sue Delbruck, AD Hershey and Salvador
level. Bacteria and their plasmids, yeasts and viruses Curia
1972 Gerald Edelman and Rodney Porter
arc mutinely employed as vectors in recombinant
1975 David Baltimore, Renato Dulbeceo and
DNA technology They have made available Howard M Tcmin
precious information and powerful techniques for 1976 S Baruch, Blumbcig and Garleton
genetic manipulation and molecular engineering. Gajdusck
They need to be used wi =,dy and well for the benefit 1976 W Arbcr, D Nathans and HO Smith
1960 Baiuj Benacexraf, Jean Dausset and George
of all Irvi ng beings.
SnelJ
The number of Nobet laureates in Medicine 1984 Niels Jerne, Cesar Milstem and Geotges
and Physiology awarded the prize for their work Kohler
in m icrobit>logy, lifted below, is evidence of the 1987 Susumu Tonegawa
positive contribution made to human health by the 1989 J. Michael Hi shun and E Vannus
Science of microbiology. 1996 Paul Doherty ana Rolf Zinkemagel
1997 Stanley Pmsiner

Furrher Reading
BtfliOtnaf B et h!. I960. A ffistary of Bacteriology and Jmmunotogy, London; William Heinemann.
Clark F F 1% l. Piuo&er Micmbinfvgrm of America. Madi fon: Univemiy ol’WLstoriiin Pitb.
Collird P 1976. The velopmenr of Micro! <iology, Cambridge Uruversny Pms.
deKruiFP 1958. Mrawbe TJunutesL London: Hutchison.
Ftmer WD 1970. A History ofMniicil Bacteriology and Immunology, London; Cm and Wyman.
Leehevdier HA and M Solotorovsky 1974. Three Centuries' ofMicrobiology. iJovtr Publications,
Marquardt M 19S1. Aiti/ Ehrlich- York bchuman.
l^riFh HJ i960. \Tictofywiih ViLtihes - The. Story of Immunisation, London: Livingstone.
VaUery Rodot R 1948. The i.:fe of Pasteur iru=:. by RL Devonshire. London: Constabbe.
Wuersnn AP Mid L Wtfjtinson 1976. An introduction fa the History of Virology. London: Cambridge University Pkeas.
Williams G i960. Vrruj fJurtBfr#. Lotidon: Hutchi'ion.

C o p y rig h te d m aterial
 Morphology and Physiology
of Bacteria

MLcroorganisme arc a heterogeneous group of

several distinct clashes o f living beings.. Thejr were
originally classified under the plant and animal
kingdoms. As this proved unsatisfactory. they were
classified under a third kingdom, the proftsra, Based
on differences in cellular organisation and
biochemistry, the kingdom protista has been divided
into two groups prokaryotes and eukaryotes.
Bacteria and blue green algae are prokaryotes,
while furtgi, Other algae, dim e moulds and
protozoa arc eukaryotes.
Bacteria are prokaryotic microorganisms that
do not contain chlorophyll. They are unicellular
and do not show true branching, except in the so-
called 'higher bacteria' (Actinomyccrales).
'Hhe unit of measurement used in bacteriology is
the micron (micrometre, fim)
1 micron (p) or micrometre (pm) - one
thousandth o f a millimetre.
1 millimicron (mp) or nanometre [nm) = one
thousandth, n: a micron o r one millionth of a
millimetre.
1 Angstrom unit (A) ■ one tenth of a nanometre.
The limit of resolution with the unaided eye is
about 200 microns. Bacteria, being much smaller,
can be visualised only under magnification. Bacteria
of medical importance generally measure 0.2—1,5
pm in diamerer and about 3 -5 in length.

Nucleus
Nuclear n-wmbrurte Absent Present
Nueleolut Absent Present
1Jeoxyrihoruiikoproicin Absent Presenr
Chromosome One (circular) Merc than one (linear)
Mtnorie division Absent Present
CyropUsm
Cytopiumie streaming Absent Present
Pinocyiosis Absent Presenr
Mitochondria Absent Present
Lysoiomej Absent Present
Golgi apparatus Absent Present
Endoplasmic reticulum Absent Present
Chemical oumposition
Strict Absent Present
Muramn? acid Present Absent

C o p y rig h te d m aterial
 fi * 1e?k:i&o o (i of M .f'o b ia ogy ►
M ic r o s c o p e
The mcjrpht]]^ 1 study of bacteria requ:res the
use of microscopes. Microscopy lias come .l long
way >ince Leeuwcnbiick firsr observed bacteria over
three hundred years ago using hand-ground lenses.
The following types of microscopes are employed
now,
Uplicul or lijjht ihionUCOpfi: Baderi.i may
he examined under the compound microscope,
citherin the Living state or after fixation and staining.
Examination of wet films or 'hanging drops"
indicates the shape, arrangement, motility and
approximate - :. m of the cells. But due to lack of
contrast, derails cannot be appreciated.
P h ase c o n tra s t m icro sco p y : improves the
contrast and makes evident the structures within
cells that differ in thickness or refractive index. Als:
the differences in refractive index between bacterial
cells and the surrounding medium make them
clearly visible. Retardation, hy ,i fraction of a
wavclengrh, of the rays of light tliat pass through
the object, compared tu the rays passing through
the Surrounding medium, produces “phase'
differences between rhe two types of rays. In the
phase contrast microscope, 'phase' differences are
converted :nto differences in intensity of light,
producing Light and dark contrast in the Linage.
D a rk field / Duck, g ro u n d m ic ro s c o p e :
Another method of improving the contrast is the
dark field (dark ground) microscope in which
reflected light is used instead of the transmitted
light used in the ordinary microscope.The essential
parr of the dark field microscope is the dark Held
condenser wh:h a central circular stop, which
illuminates the object with a cone of light, without
letnng any ray of Lujht to fall directly on the
objective lens. Light rays falling on the object are
reflected or scattered on to the objective lens, with
the result that the object appears se]f-lumi"iOUS
against a dark background. The contrast gives an
illusion of increased resolution, so that very slender
organisms, such as spirochetes, nor visible under
ordinary ilium inar.on, can be clearly seen under the
dark field microscope.
Thc resolving power of the Light microscope is
Limited by the wavelength of light- In order to be
seen and delineated (resolved), an object has to hive
a size of approximately half the wavelength of the
light used. With edible light, using the best optical
systems, the limit of resolution is about dOO nm. Jr
light of shorter wavelength is employed, as in the
ultraviolet microscope, the resolving power can be
proportionately extended,
] wO specialised types of microscopes arc 1) the
interference microscope which not only reveals cell
organelles but also enables [quantitative
measurements of the chemical constituents of cells
such as lipids, proteins and nucleic acids, and 2)
the polarisation m icmscope wh ich enables the study
of intracellular structures using differences In
birefringence.
E le c tr o n imio r o s a o p e In the electron
m: croscope, a beam of electrons is employed instead
of the beam of light used in the optical microscope.
The electron beam is focused hy circular
electromagnets, which are analogous to the lenses
in the light microscope. The object which is held
in the path of the beam scatters the electron; and
produces an image which is focused on a fluorescent
viewing screen. As the wavelength of electrons used
is approximately 0,005 run, as compared to 500 nm
with visible lighr, rhe resolving pow er of the
electron microscopes should be th e o re tica lly
100,000 times that of light microscopes but in
practice* the resolving power is about 0.1 nm.
I he technique of shadow-casting with vaporised
heavy metals has made possible pictures with good
contrast and three-dimensional effect. Another
valuable technique in studying fine structure is
negative staining with phosphotungstic acid.
Gas molecules scatter electrons, and it i;
therefore necessary to examine the object in a
vacuum. Hence, only dead and dried objects can be
exammed m the electron micntHonpc-This may lead
C o p y rig h te d m aterial
to considerable di^tmtioA in cell morphology. A
method introduced to overcome this disadvantage
La freeze-etching, invoking the deep-freezing of
specim ens in a liquid gas and the subsequent
formation of carbon-platinum replicas of the
material. Since SU^h fitwen ixlls may remain viable,
it is claimed that Ireeze-erehing enables the study
of cellular ultraHtnjctuie ay it appears in the living
state. The recent development of very high voltage
electron microscopes may render possible the
eventual examination of live objects. The scanning
electron microscope is a useful innovation which
permits the study of cell surfaces with greater
contrast and higher resolution than with the
shadow-casting technique.
Live bacteri a do not show much strut tu ral deta iI
under the light microscope due to lack of contrast.
Hence iris customary rouse staining techniq ues to
produce colour contrast. Bacteria may be stained
in the living state, but this type of staining is
employed only for special purposes. Routine
meiln.ids for staining of bacteria involve drying and
fixing smears, procedures that kill them. Bacteria
have an affinity for basic dyes due to the acidic
nature o f their protoplasm. The following are
staining tech n iq u es co m m o n ly used in
bacteriology.
Dyes such as methylene blue or
basic fuchsin are used far simple staining. I ILev
provide colour contrast, but impart Tin- same colour
to all bscteria.
H ere, bacteria are mixed
with dyes such as Indian ink or nigrosin chat provide
a uniformly coloured background agamst which the
unstained bacteria stand out in contrast. This is
particularly useful in the demonstration of bacterial
capsules which do not take simple stains. Very
slender bacteria such as spirochetes that arc nor
demonstrable by simple staining methods can be
viewed by negative staining.
Cells and structures
too rhin to be seen under the ordinary microscope
may be rendered visible if they arc thickened by
impregnation ofsilveror the surface. Such methods
are used for fbe demonstration of spirochetes and
bacterial flagella.
These stains imp.m
different colours to different bacteria or bacterial
Structures. The two most widely used differencial
stains are the Gram stain and the acid fast stain.
The Cram jfam was originally devised by the
hit.tologi.st Christian Gram (l HIM) as a method of
staining bacteria in tissues. The staining technique
consists of four steps:
1. primary staining with a paranosaniiine dvc such
as crystal violet, methyl violet or gentian violet;
2. application of a dilute solution of iodine;
3. decolourisariun with an organic solvent such as
ethanol, acetone or aniline;
i conn [e retaining with a dye o f contrasting Colour,
such as carhol fuch&Ln, safranine or neutral red.
The Gram stain differentiates bacteria into two
hroad groups. Gram po^irive bacteria are those that
resist dceolourisatioti and retain the primary stain,
pea ring violet. Gram negative bacteria are
decolourised by organic solvents and, therefore. Cake
the counters to, in, appearing ted. The exact
mechanism of the Gram reaction is not understood!.
The Gram positive cells have a more acidic
protoplasm, which may account for their retaining
the basic primary dye more strongly than the Cram
negative bacteria. Decolauruation is not an all-or-
none phenomenon. Even Gram ;>ositive cells may
he decolourised by prolonged treatment with the
organic solvent. Conversely, inadequate
decolourise inn may cause all cells to appear Gram
positive, T’hc Gram reaction may be related to the
permeability of the bacterial cell wall and
cytoplasmic membrane to the dye-iodine complex,
the < iram negative, hut not the Gram positive cells,
permitting the outflow of the complex during
dccolorisation. The Gram positive bacteria become
Cram negative when the cell wall is damaged.
Gram staining is an essential procedure used
C o p y rig h te d m aterial
in the identification of buterii and frequently is
the only method required for studying their
morphology. Gram reactivity is of considerable
importance as the Gram positive and negative
bacteria differ rn>t merely in staining uliaranten stiiis
and in smjerure but also in several other properties
such as growth requirements, susceptibility to
antibiotics and pathogenkiev-
Tbe ,icid fast stain was discovered by Ehrlich,
who found that alter staining with aniline dyes,
tubercle bacilli resist decolour! sat ion with acids.
The method, as modified by Zicbl and Neclsen, is
in common use now. The smear is stained by a
strong solution of tarhol fuchsin with tbe
application of heat. It is then decolourised with
20 per cent sulphuric arid and enunterstained with
a contrasting dye such as methylene blue. "Hie acid
fast bacteria retain the fuchsin (red) colour, while
the others take the ccHinterstain. Acid fastness has
heen ascribed to the high content and variety of
lipids, fatty acids and higher alcohols found in
tubercle bacilli. A lipid peculiar to acid fast bacilli,
■i high molecular weight hydmjfy acid wan
containing cartway] groups (mycotic acid) is acid
fast in the free state. Acid fastness is not a property
of lipids alone hut depends also on the integrity of
the Cell wall.
Depending on their shape, bacteria are classified
into several varieties (Fig. 2.1):
1, Cocci (from kokkot meaning berry) are spherical
or oval cells.
2, Bacilli (from bacuhtf meaning rod) ate tod
shaped cells.
2. Vibrios are comma shaped, curved rods, and
derive the name from their characteristic
vibratory motility,
are rigid spiral forms.
5. Spirochetes (from uprira meaning coil and charte
m eaning hair) are tlexuous spiral forms..
6 . Actitiom y eett s are branching filamentous
bacteria, so called because of a fancied
resemblance to the radiating rays of the sun when
seen in tissue lesions (from aefis meaning ray
and mykics meaning fungus).
7. Mycoplasmis are bacteria rhit are cell wall
deficient and hence do out pOfsess a stable
morphology; They occur as r<>und or oval bodies
and as inter I,wing riLirncn;>. \Vben cell wall
synthesis becomes defective, either
spontaneously or as a result of drugs like
penicillin, bacteria lose their distinctive shape.
Such cells are called protoplasts, sphenopla&ts
or L forms.
Bacteria sometimes show characteristic cellular
arrangement or grouping (Fig- 2.2!. Thus, cocci
may be arranged in pairs (diplococci), chains
{streptococci^ groups of four .tetrads or eight
(sareini), or is grape-like dusters (staphylococciX
Some bacilli too may be arranged in chains
(stneptobacilli). Others are arranged at angles to
each other, presenting a cuneiform o- Chinese letter
pattern (curynebacieria). The type of cellular
arrangement Is determined by the plane through
which biraty fission takes place and by the tendency
of the daughter cells to remain attached even after
division.
• ’
C o p y rig h te d m aterial
 r : t; -; . ;

J *
V
4
1

Fig. 2.2 AiTHitvgwnfnl c? r-:^el: 1. plnptncpecl Taj, M of : t, l»dRI In cluster

2. ppunsacocrl 3. pracMd 4 mfringococcl 2. bieI i ii tin Inn a into m : 3.. ti pIs' cc-I
5. MWtawto HjM M k L flMKp W i p i (K. pK-LT.HflJlJf ?j
7- tardna ELrTc~h>tocwel

H> 2i : : : ” m e in i '.z'.'.zzd '•* I::lal ci'I :

I. Cipaute 2; Pill 3,Gutter 1. ESii’hlrn
:: :«;a i i. &mi i 7. *3 . . :.* l .
Fig. 3_4 tatn g^ T O ttt curved : scc-if Ii : 1. vibrio mm a Ftegtite i Fn Qtefcutec to. W h m h
2 . spirilla 3. spirochetes I I . J 11 ■■ ': '- - 12 I ■ 1

C o p y rig h te d m aterial

Fig. 2.5 shows the stroctuie of an idealised bacterial
Celt The outer layer of lcII envelope consists dt
two components- ;l rigid cd] wall and beneath it ;l
cytoplasmic or plasma membrane, The cell envelope
encloses the protoplasm, comprising the cytoplasm,
cytoplasmic inclusions such ;l5 ribosomes- and
mesosomes, granules, vacuoles and rhr nuclear body
Besides these tssenEdl components, some bacteria
imav I'OFse-KF additional structures. The cell may he
enclosed in a viscid layer, which may he .1 loose
slime layer, cmu ja d ie d ,i> a capsule. Some bacteria
carry filamentous appendages protruding from the
cell surface - the flagclht whiL'h ? it nrg.ins ot
Inedmotion and the llmbrne wllic] 1 appear to he
organs for adhesion.
The tell wall accounts for the
shape of the bacterial ll-11 and confers on it rigidity
and ductil ity. T'he cell wall ca nnot he see n by t\irert
light microscopy arid does; not stain with sii[i]ilc
Stains. Ir may he demonstrated he phsmolynii.
W hen placed in a hypertonic Solution, the
cytoplasm loses water by osmosis and shrinks, while
the cell wall retains its original shape and siv.e
{bacterial ghost). The cell wall may also be
demonstrated hv micinditlectian, reaction with
specific antibody, mechanical rupture of the cell,
differential sraining procedures or by electron
microscopy. Bacterial cell walls are about 1CF-25
nm thick and account for about 20-50 per cent of
the dry weight of the cells. Chemically the cell
wall is t orn posed (it rnuccipeptitfe (pcptidoglyciTi
cn murein) scaffolding formed by N icetyl
glucosamine and K ai elyl niuramic acid molecules
alternating in chains, which arc cross!inked hy
Thickness
Variety of aminoacids
Aromatic and sulphur containing aminoacids
Lipids
Tekhoic acid
peptide thains (big- 2.h), The interstices ot this
vcaflfoidmg contain other chemicals, varying in the
different species. In general, tlic waits of the Gnarrs
positive bacteria have si triplet chemical nature than
those of Gram negative bacteria (Table 2 . 1 ) . The
cell wall carries bacterial antigens that are
important in virulence and immunity.
The lipopalysacchdrida (LPS) pretext on the
eel! walls of Gram negative bacteria account for
[heir cndotoxic activity and O antigen specificity,
T hey were formerly known as the Boivin antigen.
T he LPS consists ot three regions. Region T is the
polysaccharide porcion determining rbe O antigen
specificity. Region II is the core polysaccharide,
Ur-gioit II Ms the glycolipid portion (lipid A) and
i* responsible for the cndotoxic activities, that is,
pyrogeiiicity, lethal cftcct, tissue necrosis,
,mti complementary activity, R cell mitogenicity,
immunoadjuvutt property and antitumour activity.
The outermost layer of Gram negative bacterial
cell wall is called rhe outer membrane, which
contains various proteins known as outer membrane
proteins (OM F). Among these are porins which
form transmembrane pores [bat serve a* diffusion
channels tor small molecules- They also serve as
specific receptor* for some bacteriophages.
Cell wall synthesis may be inhibited by many
fecTtKre. Lysozyme, an eir/yme normally present in
many tissue fluids, lyses susceptible bacteria by
split Ling rbe cell wall mucopeptide linkages. When
lysozyme acts on a Gram positive bacterium in a
hypertonic solution, a protoplast is formed,
consisting of the cytoplasmic membrane and its
contents. With Gram negative bacteria, the result
is a sphenrpla&r which differs fnSili the protoplast
Thicker Thinner
Few Several
Absent Present
Absent or scant Present
Present Absent
C o p y rig h te d m aterial

in that some cell wall material it retained.
Protoplasts and spheroplasts are spherical,
regardless of the ordinal shape of the bacterium.
Cell wall deficient forms of bacteria may probably
hive a rale in the persistence of certain chronic
infections such as pyelonephritis.
The cytoplasmic
(plisitiu) membrane is a thin (5-10 nm) layer lining
the inner surface of the cell wall and separating .it
from the cytoplasm. It acts as a sennpermeable
membrane controlling the inflow and outflow of
metabolites to and from the protoplasm, Passage
through the membrane is not solely a function of
the molecular size of the particles bait depends, in
many cases, on the presence in the membrane of
specific enzymes (pemieaites.f. Electron microscopy
shows the presence of three layers- constituting a
'unit membrane' s tru ctu re . Chemicatly, the
membrane consists of lipoprotein with small
amounts o f carbohydrate. Sterols are sb sn u f except
in mycoplasma.
The bacterid cytoplasm is a Colloidal
system uf a variety o f organic and inorganic solutes
in a viscous watery solution. It differs from
eukaryotic cytoplasm in not exhibiting internal
mobility (protoplasmic streaming) and in, the
absence of endoplasmic reticulum or mitochondria.
The cytoplasm stains uniformly with basic dyes in
young cultures hut becomes increasingly granular
wit'll ige. The cytoplasm contains rihnsomcs,
mesosOiTies, inclusions and vacuoles,
are centres of protehi synthesis. They
are slightly smallci than the ribosomes of eukaryotic
Cell'S (sedimentation constant 70 S) and are seen
integrated ill linear strands of mRNA to form
polysomes,
(chon droids] are vesicular,
convoluted or mulrilaminated structures formed as
Liiyaginations of the plasma membrane into the
cytoplasm. They arc ivu>rc prominent in Gram
positive bacteria. They are the principal sites of
respiratory enzymes in bacteria and arc analogous
to the mitochondria of eukaryotes. M cstW /no arc
■often Seen in relation to the nuclear body and the
site o f syn thesis of CTOSS wall septa, Suggesting that
they coordinate nuclear and cytoplasmic division
during binary fisrion.
may be of
various types, the chief of which arc volutin,
polysaccharide, lipid and crystals. They are
characteristic for different species and depend on
the age and condition of the culture. Volutin
granules { mctachrMMfJC0T B ibet-E m si granules)
are highly refractive, strongly basophilic bodies
consisting of poly me laplmsp hate. They appear
reddish when stained with polychrome methylene
blue or tohiidlne blue (inetitchmmasia). Special]
staining reebniquCS Such as Albert’s or Neisser’s
demonstrate the granules more clearly. Volutin
granules are characteristically present in diphtheria
bacilli. Their function is uncertain. They have been
considered to rep resent a reserve of energy and
phosphate for cell metabolism but they are most
frequent in cells grown under conditions of
nutritional deficiency and tend to disappear when
the deficient nutrients are supplied. Polysaccharide
granules may be demonstrated by staining with
C o p y rig h te d m aterial
14 * Textbook ol Microti, blopy *
iodine, and lipid inclusions wi^h fat soluble dyes
such as Sudan hlack. They appear to be storage
products. Vacuoles ire fluid-containing cavities
separated from the cytoplasm bv a membrane. Their
function and significance arc uncertain.
N ucleus: Bacien^il nuclei can be demonstrated
by and or ri iMjnuclease hydrolysis and subsequent
staining for nuclear material-They maybe wen by
electron microscopy. They appear as oval or
elongated bodies generally one per cell. Some cells
may possess two or more nuclear bodies due to
asynchrony between nuclear and cytoplasmic
division.
Racferlal nuclei have no nuclear membrane or
nucleolus. The nuclear deoxyribonucleic acid
[DNA) is not associated with basic protein. The
genome consists of a single molecule nf double
stranded DNA arranged in the form of a circle,
which may upen under certain conditions to form
a long chain, about 1 mm in length. The bacterial
chromosome is haploid and replicates hy simple
fission instead of by mitosis as in higher ceiJs. The
differences between the nuclei of bacteria and higher
organisms form the main basis for classifying rhem
as prokaryotes and eukaryotes.
Bacteria may possess extrinuclear genetic
elements consisting of DNA. These cytoplasmic
earners of generic informatfon are termed pfasm/ifs
or epfsomes [see Chapter 6). Besides being
transmitted to daughter cells during binary fission,
they may be transferred from one bacterium to
another cither through conjugation or the agency
of bacteriophage They are not esscnriid for the
life of the cell they inhabit but may confer on it
certain properties like tux igeni city and drug
resistance which may constitute a survival
ailv.Lnt uul"-
Slim u la y e r an d c a p s u le : Many bacteria
secrete a viscid mate dial around the ceil surface.
When this is organised into a sharpie defined
structure, as in the pneumococcus, it is known as
the capsule. When it is a loose undemarcated
secretion, as in leuconostoc, ir is called! the slime
foyer- Capsules too thin to be seen under the light
microscopes are called mrLrrocajuufer. The slime
is generally, but not invariably, polysaccharide (for
example pneumococcus] or polypeptide [for
example anthrax bacillus! In nature. Some bacteria
may have both a capsule and a slime layer (for
example. Streptococcus s&lir&rius). Bacteria
secreting large amounts of slime produce mucoid
growth on agar, which is of a stringy consistency
when touched with the loop.
Slime has little affinity for ba>ic dves and is not
visible in Gram stained smears. Special capsule
staining techniques are available, usually employing
copper salts as mordants. Capsules may be readily
demonstrated by negative staining in wet films with
India ink* when they are seen as dear halos around
the bacteria, agyiin&ta black background [Fig, 2.7).
Capsular material is antigenic and may be
demonstrated by serological methods. When a
suspension of a capsulated bacterium is mixed with
its specific anti-capsular serum and examined under
the microscope, the capsule becomes very
prominent and appears 'swollen' due to an increase
in its reffactmty. This capsule swelling or Qpel/ung
reaction, described by Ncufcld (1902) was widely
employed for the typing of pneumococci in the pre-
sidphonamide days when lobar pneumonia used to
be treated with specific anticapsuku sera. Capsules
protect bacteria from deleterious agents such asiyuc
enzymes found in nature. They also contribute to
the virulence of pathogenic bacteria by inhibiting
phagocytosis. Loss of the capsule by mutation may
tender rhe bacterium a vim lent. Repeated
subcultures in vitro lead to the Joss of capsule and
.ilso of virulence.
I" 1age) Ia: Motile bacteria, except spirochetes,
possess one or more unbranched, long, sinuous
filaments called flagella, which arc the organs of
locomotion. Each flagellum consists of three distinct
parts, the filament, the hook and the basal body.
The filament is external to the cell and connected
to the hook it the cell surface.
The hook-basal body portion is embedded in
C o p y rig h te d m aterial
the cell envelope. The hook. and basal body arc
antigcnicilly different. Mechanical detachment of
the filament does not impair the viability of the
cell. The flagella are 3 -2 0 pm long and arc of
uniform diameter (0.01-0.013 pm) and terminate
in a square dp. The wavelength and thickness of
the filament are characteristic of each species but
Rome bacteria exhibit hipliclty, that is, they have
flagella of two different wavelengths (Fig. 2,8),
Flagella aic made up of a protein (flagellin) similar
to keratin or myosin- Though flagella of different
genera of bacteria have the same chemical
composition, they art ant [genic ally Jit ft rent.
Flagellar antigens induce specific antibodies in high
titres. Flagellar antibodies arc not protective hut
are useful in serndi agnosia
The presence or absence of fkgclla and their
number and arrangement arc characteristic of
different genera of bacrerii (Fig, 2.9), Flagella may
he arranged all round (he ceil {peritridhnus) as in
typhoid bacilli, or situated at one or both ends of
the cell (polar). Polar flagella may be single
(monotrichous) as in cholera vibrios, in tufts
(lophotrichous) as in spirilla or with flagella at
both poles (amphitrichous).
Flagella arc less than 0-02 iim in thickness and
hence beyond the limit of resolution of the light
microscope. They' may, in some instances, be seen
under dark ground illumination. They can be
visualised by special staining techniques in which
their thickness is increased by mordanting, or by
electron microscopy (Fig. 2.10). Due to the
difficulty of demonstrating flagella dhecdyhtheir
presence is usually inferred from the motility of
bacteria. Motility can be observed by noting the
spreading type of growth on a sent isolid agar
medium. Under the microscope^ active motility has
to be differentiated Ifom the passive movements of
the ccllsT cither due to air currents or due to
Brownian movement. Bacterial motility may range
from the slow'stately' motion ofpcriirichatc bacteria
(for example. Bacillus) to the darting movement of
polar flagellated vibrios. The cholera vihriu may
move as fast as 200 pm per second.
Some Gram negative bacilli carry very
fine, hair-like surface appendages called fimbriae
or pili. They are shorter and thinner than flagella
(about 0.5 pm long and less than lOnm thick) and
project from the cell surface as straight filaments.
At least eight morphological types of pili are
known, classifiable as either common or sea; pili
on the basis of their fimedon. Pili comprise self-
aggregadng monomers of pilln. They originate in
the cell membrane, Fimbriae can be seen only under
the electron microscope. They are unrelated to
motility and are found on motile as well as
nan motile cells, lltcy arc best developed in freshly
isolated stratus and in liquid Cultures.They tend IO
disappear following subcultures on solid media.
Fimbriac function ns organs of adhesion, helping
the cells to adhere firmly to particles of various
kinds. This property may serve to anchor the
bacteria in nutritionally favourable micro
environments. Fimbriated bacteria form surface
pellicles in Liquid media. Many Fimbriated cells (for
example Escherichia, Klebsiella) agglutinate ted
C o p y rig h te d m aterial
blood Cells o f guinea pig$, fiiwl, horsey and piyv
strongly, human and sheep cells weakly, and ox
cells scarcely at alt
Hemagglutination p r id e s a simple method for
detecting the presence of such fimbriae. The
hemagglutination -v specifically inhibited by D-
rrurnose (mannose sensitive).
fimbriae are antigenic. As member* of different
genera may possess rhe same fimbrial antigen, it is
necessary to ensure that the bacterial antigen*
employed for serological tests and preparation of
antisera are devoid ot fimbriae.
A special type of fimbria arc the sex: pili. These
arc longer and fewer in number than other fimbriae.
They are found or 'male' bacteria and help iti the
attachment of those cells to 'female' bacteria,

forming hollow conjugation Tubes through which,

it is assumed, genetic material is transferred from
[be donor to the recipient cell. IV: are classified
into different types (for example, K 1) based on
susceptibility to specific bacteriophages.
Some bacteria, particularly members of
the gene ra 11aci llus and C Eostridium have the ibili ry
to form highly lerisLant resting stages. called spores.
Each bacterium forms one spore, which on
germi.nari.ou forms a single vegetative cell.
Speculation in bacteria, therefore. is not a method
of reproduction As bacterial spares are farmed
inside the parent cell, they are called endospoies.
While the exact stimulus for Kpomlation is not
known, it occurs after a period of vegetative growth
and ls presumed to be related to the depletion of
otogenous nutrients, Sporulition is initiated by the
appearance of a clear area, usually near one end of
the cell, which irr;LduaUv becomes more opaque tn
form the 'fbrespure’. The fully dndciped spore has

C o p y rig h te d m aterial

at its core the nuclear body, surrounded by the spore
wall, a delicate membrane from which the celt wall
of the future vegetative bacterium will develop, Outside
this is the thick spore oxtcc, which in mm is enclosed
by a multilayered tough spore coat. Some spores have
an additional outer covering called exosportunn, which
may have distinctive ridges and grooves (Fig, 2.11).
New antigens appear on speculation.
Young s[)t>res are seen attached to the parent
cell. The shape anti position of the spore and its
si ye relative to the parent cell ure species
characteristics. Spores may he central
(ptJOatoriai), terminal or suhterminal. They may he
oval or spherical.' fhey in ay or may rot distend the
bacillary body (Fig. 2.12).
Bacterial spores constitute some of the most
resistant forms of life. ITicy may remain viable for
centuries. 'I’hey are extremely resistant to desiccation
and relatively so to chemicals and heat. Though
some spores may resist boiling for prolonged
periods, spores of all medically important species
are destroyed by autoclaving at L20 °C for 15
minutes. Methods of sterilisation and disinfection
ifcrsgj of w m m * * #7
should ensure that spores also are destroyed.
Spoliation helps bacteria survive for long periods
under unfavourable environments.
When transferred to conditions conducive for
growth, spores germinate. The spore loses its
refractiUiy and swells. The spore wall is shed and
the germ cell appears hy rapturing the spore coat
nid elongates to form the vegetative bacterium,
Spores maybe seen in unstained preparation as
refrac-tile bodies. The fcrespore stains intensely, but
once the spore envelope ls laid down, the spore
does not stain rcadiEy. Spores appear as unstained
areas in f Iram stained preparations, hut being more
acid fast than the vegetative cells, thev can Ik: stained
by a modification of the Ziehl-Ncelscn technique.
Some species of bacteria eshihit great variation in
the shape and size of individual cells.This is known
as pleo morph ism. Certain species (for example,
plague bacillus, gonococcus) show swollen and
aberrant forms in ageing cultures, especially in the
presence of high salt concentration. These are
known an involution forms. Many of the cells may
C o p y rig h te d m aterial
 '
be nonviable. Plcomorphism anti involution Forms
are often due to defective cell wall synthesis.
Involution forms may also develop due m the activity
of aiitolytie enzymes.
Kleioeberger-Nobel, Studying cultures
of Sfirptiihzoffus moniliformis, observed swollen
cells and other aberrant morphological forms and
named them L forms, after Lister Institute, London,
where the observation was made, L forms arc seen
in several species of bacteria, developing either
spontaneously nr in the presence of penicillin nr
other agents that interfere with cell wall synthesis.
L forms may be unstable m thar eke morphological
abnormality is maintained on|v in the presence of
penicillin or other inducing agents, or stable, when
the aherrant form becomes rhe permanent feature
of the strain and is retained in serial subcultures. L
forms resemble mycoplasma in several wavs,
including morphology, type of growth on agar and
filter-ability. It is possible that myeoplasmis
iq iresent stable I, forms of as vet kmiden ti hied pane nr
bacteria.
Bacteria divide by binary fission. When a bacterial
cell reaches a certain size, it divides to form two
daughter cells. Nuclear division precedes cell
division and, therefore, in a growing population,
many cells carrying two tiuck-ar bodies can be seen.
The cel] divides by a constrictive or pinching
process, or by the ingrowth of a transverse septum
across the cell. In some species, the daughter cells
may remain partially attached alter division.
The interval of time between two cell divisions,
or the time required for a bacterium to give rise to
two daughter cells under optimum conditions, is
known as the genera cion time or population
doubling time- In coliform bacilli and many other
medically important bacteria, the generation time
is about JO minutes. Some bacteria are slow-
growings the generation time in tubercle bacilli is
about 20 hours and in lepra bacilli as long as about
20 days. As bacteria reproduce so rapidly and by
geometric progression, a single hacterial ceil can
theoretically give rise to 10*1 progeny in 24 houn,
wi rh a mass of approximately 4,000 tonnes! 1n actual
practice, when bacteria arc grown in a vessel of
liquid medium (batch culture . multiplication is
arrested after a few cell divisions due to depletion of
nutrients or accumulation of rnxic products. By the
use of special devices for replenishing nutrients and
removing bacterial cells (chcmostat or turbidisrat), it
is possible to maintain con dn llous cutaije of bacteria
tor industrial or research purpose#. When pathogenic
bacteria multiply in host tissues, the situation may be
intermediate between a IVLtch coin irv ,i ru L.la m rin u<uis
culture; the source of nutrients may be inexhaustible
but the parasite has to contend with the defence
mechanisms of the body Bacteria growing on solid
lililt sot
C o p y rig h te d m aterial
 h Mo'phoiogv and Physiotofly td Bacteria »
media form colonics. Each colony represents a done
of ccILh derived from A single parent cell. In Iil: uni
media, growth i-: diffuse.
Bacteril growth may be considered at two lcvds,
increase in the size of the individual cell and increase
:n the number of cells. The former is ordinarily limited
and when the critical s i* is reached* the cell divides,
except when cetl division is inhibited bv suhstaners
like penicillin or ac rifbmnc or bygrowth i it magn&i um
deficient media. Crowd 1 in numbers can be studied
hy bacterial counts. Twi] types of hactipnd counts can
be made— total count and viable count.
'Hie total count gives the total number of cells
m the sample* irrespective of whether they are living
or not. It can he obtained hy
1. direct counting under the microscope u-iing
counting chamber?,
2. counting in an electronic device as in the Coulter
counter,
3. direct counting m-ing st.lined smears prepared
by spreading a known volume of the culture
aver a measured area of a slide,
4 . comparing relative numbers in smears ofthe culture
mixed with known numbers of other cells,
5. by opacity measurements using an absnrptio
meter or nephalomcter,
6. by separating the cells by centrifugation or
filtration and measuring their wet or dry weight,
and
7. chemical assay of celt components such as
nitrogen.
The viable count measures the number of living
cells, that is, cells, capable of multiplication. Viable
counts are- obtained by dilution or plating methods.
In the dilution method, the suspension is diluted
to a point beyond which unit quantities do not yield
growth when modulated into suuable liquid media
(extinction). Several tubes are uioculated with
varying dilutions and the viable count calculated
statistically ftom the number of tubes showing
growth. The method docs not give accurate values
but is used widely in water bacteriology for estimation
of [lie 'presumptive eoliform count' in drinking water.
19
In the plating method, appropriate dilutions arc
inoculated on solid media, either on the surface of
plates or as pour plates. The number of colon ics that
develop after incubation giyes an estimate of the viable
count. The method commonly employed is that
described by Miles and Misra [193E) in which serial
dilutions are dropped on the surface of dried plates
and colony counts olrtiiined.
& a c i b r i a l G r o w t h C u rv e
When a bacterium h seeded into a suiruble liquid
medium and incubated, its growth follows a dcfinirc
course, Ifbactcrial counts are made at intervals after
inoculation and plotted in relation to rime, a growth
curve is oh Mined [Fig- 2 . 13 ). The curve shows the
following phases:
Lug ])liase: Immediately following the seeding
of a culture medium, there is no appreciable
increase in numhers, though there mav be an
increase in the size of the cells. This initial period
is the time required for adaptation to the new
environment, during whi-b the necessary enzymes
and metabolic intermediates are built up in adequate
quantities for multiplication to proceed. The
dura non of the lag phase varies with the species,
size of inoculum, nature of culture medium and
environmental factors such as temperature.
L o g Uogfl rithftiic) or exponential! phase:
Following the Lag phase, the cells start dividing
and thi ir numbers increase exponentially or by
geometric progression wbh time. If the logarithm
of the viable count is plotted a^unsi nme, a straijfot
line will he obtained.
S tatio n ary phase; A fter a varying period of
exponential growth, cel) division stops due to
depletion of nutrients and accumulation of toxic
products. The number of progeny cells formed is
just enough to replace the number of cells that die.
The viable count remains stationary as an
equilibrium exists between the dying cells and the
newly Formed cells.
E^luiso til dudiiiu: Tim is the phase when the
population decreases due to cell death. Besides
C o p y rig h te d m aterial

nutritional exhaustion and toxic accumulation, cell
death may also be caused by imtolytic cniym cs
W hen the total count is plotted, it parallels the
viable count up to the Stationary phase but it
ContiHire* steadily without any phase o f decline.
W ith autolytic haercria, even the total cnun t shows
a phase o f decline.
The various stages of the grow th Curve are
associated with morphological and physiological
alterations o f the cells. Bacteria have the maximum
cell size towards the end o f the lag phase. In the
log phase, cells ate smaller and stain uniformly. In
the stationary phase, cells frequently are Gram
variable and show irregular staining due to the
p resen ce of in tra ce llu la r sto rag e gran u les.
Spoiulation occurs at this stage. Also, many bacteria
produce secondary m etabolic products such as
CSOfnjtins and antibiotics. Involution forms are
common in the phase o f decline.
rl'hc bacterial cell has the same general chemical
pattern as the cells of higher organism s. T h e
principal constituent o f bacterial cells is water,
which represents about SO pet cent of the total
weight. Proteins, polysaccharides, Lipids, nucleic
acids, mucopcptidcs and W r molecular weight
compounds make up the rest, bacterial metabolism
also is closely similar to the metabolism of die higher
organisms, exemplifying the ‘unity of biochemistry'.
There arc. however, some differences which are
exploited in selective toxicity and chemotherapy
Ibr growth and multiplication of bacteria, the
minimum nutritional requirements are water, a
source of carbon, a source of nitrogen and some
inorganic salts. Water is the vehicle far the entry
of dll nutrients into the cclh and lor the elimination
of all waste products. It participates in [he metabolic
reactions and also forms an integral port of the
protoplasm.
Bacteria can. be classified nutritionally, based On
the it energy requirements and on their ability to
synthesise essential metabolites, Bacteria which
derive their energy from sunlight ate called
phototrophs and those that obtain energy from
chemical reactions are called ebemorrop/rs. Bacteria
that can synthesize all their organic compounds are
called surcUTophs. Those that are unable to
synthesise their own metabolites and depend on
pr-cfomed Organic compounds are called
heterotrophs. Aurotrophs arc able to utilise
atmospheric carbon dioxide and nitrogen. They are
capable of independent existence in water and soil
and are of no medical importance, though they are
of vital concert i lii agriculture and the maintenance
of soil fertility- Heterotrophic bacteria are unable
m grow with carbon dioxide as the sole source of
carbon. The nutritional requirements o f
heterocrophs vary widely Some may require only a
single organic substance such as glucose, while
others may need a large number of different
compounds such as ami none ids, nucleotides, lipids,
carbohydrates and coenzymez.
hactcria require a supply of inorganic salts,
particularly the anions phosphate and sulphate, and
the cations sodium, potassium, magnesium, iron,
manganese and calcium. These are normally present
C o p y rig h te d m aterial
 h Morphology anti Fhy ioiogy ol BaGtoria *
in the natural environments where bacteria live but
will have to be supplied in culture media. Some
ions such as cobalt maybe needed in nacc amounts-
Suntf bacteria require certain organic
compounds in minute quantities. These are known
as growth factors or bacterial vitamins. Growth
factors are called « « n tu f when growth docs not
occur in their absence, or accessory when they
enhance growth, without being absolutely necessary
for it. In many cases, bacterial vi tarnins are identical
with the vitamins necessary for mammalian
nutrition, particularly those belonging to the B
group, thiamine, nhoflavlne, nicotinic acid,
pyridoxine, folic add and vitamin B l 2 ,
Ifa microoigaiuMO reqmringan essential growth
factor is inoculated into a medium containing an
excess of all other nutrients, its growth wii] be
proportional to the amount of the limiting substance
added. Within a certain range, the concentration
of the growth factor will bear a linear relationship
to the amount of growth of the organism. This is
the principle of microbiological assays, which
provide a very sensitive and specific method for
estimation of many aminoai.-:da and vitamins, as in
the determination of vitamin EMl using
LacfohaciWtiff h k h m m n ii.
O xygen R e q u ir e m e n t an d
M btabo m sm
Depending on the influence of oxygen on growth
and viability bacteria are divided into aerobes and
anaerobes. Aerobic bacteria require oxygen for
growth. They may be obligate aerobes like the
cbolcru vihrlcppwliieh wiLl jjriMi [inly in the presence
of oxygen, or facultative anaerobes which are
ordinarily aerobic but can also grow in the absence
of oxygen, though less abundandy. Most bacteria
of medical importance are facultative anaerobes.
Anaerobic bacteria, such as closmdia, grow in the
absence of oxygen and the obii^ate anaerobes may
even die or exposure to oxygen- Microaerophilic
bacteria are rh<i- l: that grow best in the presence of
a low oxygen tension.
21
The reason for the apparent toxicity of oxygen
for anaerobic bacteria is not wcl] understood. If
has been suggested that in the presence of oxygen,
hydrogen peroxide and other toxic per ox idea
accumulate. The enzyme catalase which splits
hydrogen peroxide is present in most aerobic
bacteria but is absent in the anaerobes. Another
reason might be that obligate anaerobes possess
essential enzymes that are active only in the reduced
state.
The influence of free oxygen is related to the
metabolic character of the bacterium. Aerobic
bactcri:i obtain their energy and intermediates only
through oxidation involving oxygen as the ultimate
hydrogen acceptor, while the anaerobes use
hydrogen acceptors other than oxygen. Facultative
anaerobes may act in both ways, In the case of
aerobes, where the ultimate electron acceptor is
atmospheric oxygen (aerobic respiration), the
carbon and energy source may be completely
Oxidised to carbon dioxide and water. Energy is
provided by the production of energy-rich
phosphite bonds and the conversion of adenosine
diphosphate (A D F) to adenorine triphosphate
(Ad P). T his process is known as oxidative
phosphorylation. Anaerobic bacteria use as electron
acceptors compounds such as nitrates or sulphates
instead of oxygen (anaerobic respiration), A more
common process in anaerobic metabolism may be
a series of oaidoreduction^ in which the carbon
and energy source acts as both the electron donor
and electron acceptor. This process is known as
fermentation and leads to the formation of several
nrgaitiu' end p n k11ll'I*: such ax .11'il4■- qnd .11. uhtiU, ax
well as of gas (carbon dioxide and hydrogen).
During the process of fermentation, energy-rich
phosphate bonds arc produced by the introduction
of organic phosphate into intermediate metabolites.
This process is known as substrate-tevel
phosphorylation. Tbe energy-rich phosphate
gtuups so formed ate used for conversion of ADF
to ATP
In determining the growth of aerobic and
C o p y rig h te d m aterial
22 ^ Tgjrtboak a1 Microtiiplggy *■
anaerobic bacien.L, what Is more important LIi-.ii:
the presence or absence of oxygen is the state of
oxidation of the environment, 'Hie oxidising or
reducing condition of a system is indicated by the
net readiness of all the components in that system
to cake up, or pare with electrons. This is known as
the oxidation—reduction \redox-' porrnrjaJ of the
system. The redox potcnti.d of a medium is best
estimated try measuring the electrical potential
difference set up between the medium and an
unattackable electrode immersed in it. This
electrode potential (E h ) can he measured in
millivolts. The more oxidised the systemHthe higher
the potential. A simpler, though less accurate,
method of measuri :ig the redo* potential is the use
of oxidation-reduction indicators such as methylene
blue, and noting the change in colour.
C arbon D io x id e
All bacteria require smalt amounts of carbon
dioxide for growth. This requirement is usually met
by the carbon dioxide present in the atmosphere,
or produced endogenously by cellular metabolism.
Some bacteria like BtuCells JtburTuS require much
higher levels of carbon dioxide (5 -10 per cent) for
growth, especially on fresh isolation (capnophilic).
T b m MSRATUB il
Bacteria vary in their requirements of temperature
for growth. For each species, there is a 'temperature
range', and growth does not occur above the
maximum or below the minimum of the range. The
temperature at which growth occurs best is known
as the "optimum temperature’, which in the case of
most pathogenic bacteria is 37 UC, Bactcrii which
grow best ar temperatures of 2 5 ^ 0 “C are called
mcsopfv/hc. All parasites of warm blooded animals
are mesophilic Within the group of mesophilic
bactcri i, some like TscLfdomcuitfs aeruginosa have
i wider range (5-43 ‘ C ), while others like the
gonococcus have a restricted range (31H39 *C).
P s y c h r t i p h i l i c bacteria are those that grow best
it temperatures below 20 aC, some of them even
growing at temperatures as low as - 7 °C. They are
soil and water saprophytes and though not of direct
medical impoctance, may cause spoilage of refrigerated
food. Another group of nonpathogenic bacteria, the
rhenTKJpfj.'.fcr, grow best at h igh temperatures, 55­
80 UC, They may cause spoilage of uiiderproce&sed
canned food- Some t her mop Kile; (like Bacf/.fus
sfcaro therm ophilus) form spores that are
exceptionally thermores istant. Extremely
thermophilic bacteria have been identified which
can grow at temperatures as high as 250 °C-
Bictma also differ in the effect of temperature on
viability. Heat is an important method for the
destructnn of microorganisms (sterilisarionh moist
beat causi ng cnagi Llatii jn ard , leriafiir.itiiin of proteins
and dry heat causing oxkktion and chaning. Moist
heat is more lethal than drv " at heat.
“ The lowest
temperature that kills a hactcrium under Standard
conditions in a given time is known as the thermal
death point. Under moist conditions most vegetative,
mesophikc bacteria have a thermal death point
between 50 and 65 and most spores between
100 and 120 *C.
At low temperatures some species die rapidly
but most survive well. Storage in the refrigerator
(3-5 “C) or the deep freeze cabinet (-3 0 to -7 0 “C)
is used for preservation of cultures. Rapid freezing
as with so Iid carbon J iodide or the use of a stabil incr
such as glycerol, minimises the death of cells on
freezing,
MOIST! RE AND D m INC
Water is an essential ingredient of bacterial
protoplasm and hence drying is lethal to cells.
However, the effect of drying varies in different
species, Some delicate bacteria like Treponema
pallidum arc highly sensitive, while others like
staphylococci withstand drying for months. Spores
arc particularly resistant to desiccation and may survive
ul the dry state for several decades. Drying in vacuum
ul the cold (fruLVCiJnfrjgoi lvophili$at3nn) is a method
for the preservation ofbacteria, viruses and manylabile
biological materials.
C o p y rig h te d m aterial

Bacteria are sensitive to variations in pH, Each
specie* hw a pH range, above or below which it
does not survive, and an optimum pH at which it
grown best. The majority of" pathogenic bacteria
grow best at neutral or slightly alkaline reaction
(pH 7.2-7.6), Some acidophilic bacteria such as
tacfobacilli grow under acidic conditions. Others,
such as the cholera vibrio, ate very sensitive t» acid,
but tolerate high degrees of alkalinity. Strong
solutions of acid or alkali (5% hydrochloric add
or sodium hydroxide) readily kill most bacteria,
though mycobacteria arc exceptionally resistant to
only on exposure to light and not when incubated
in the dark.
Bacteria am more tolerant to osmotic variation than
most other cells due to the mechanical strength o f
their cell walls. Sudden exposure to hypertonic
solutions may cause osmotic withdrawal o f water
Hid shrinkage o f protoplasm - p J a s i r i a l y s i f , T h is
OCCUR more -readily 111 G ram negative than in G ram
p o sitiv e b a c te ria . Su d d en tr a n s fe r fro m a
concentrated solution to divided water may cause
p } jtS iftO p iy $ is (excessive Osntotie im bibition leading

them, to swelling and. rupture of the cell).

Bacteria (except the phocotrophie species) grow well
in the dark. They are sensitive to ultraviolet light and
orlier radiations. Cultures die if exposed to sunlight.
Exposure to light may influence pigment production.
Phorochromogenic mycobacteria form a pigment
Though bacteria have tough cell wnlU, they may
be ruptured hy mechanical stress such as grinding
or vigorous shaking with glass beads. They may
also be disintegrated by exposure To ultrasonic
vibration.

Wlir DF 1995. Hii-.t hjL'lCfia SCQif and s-wim Annual Rev \tirrnbiol iV'.iWt.
jlld A S ichk'iUnKl IfNl. M icrrb in l M t y ilo t o fj, - rJ lU. (taliml BLrckwcU.
tiuuld GW Hid A Mum L$Bj. TV B e c t f r i a l S p o r t, Lnmtofi: Academic Pleci
VEi.jiLAG and JW Fuller IMS. M irm b itil P h v tia lo fy ,, edr. Vew ^fork Wj|fj-UiM
Sumier PY e( -I IW16. Thr Microbial WVtiJ, ^ rdn. New Jersey: Prendce-Hall.

C o p y rig h te d m ateri
 Sterilisation and Disinfection

MicrDDiganiin]!i are ubiquitous. Since they cause betbre sterilisation or disinfection, by removing soil
co n t.im j nation, infection and decay, n becomes and ocher dirt and reducing the microbial burden,
necessary to remove or destroy them from materials o iaid] ig sterilisation more eflecth'e. Decotitaniim qljn
or from areas. This is rhe object of sterilisation. refers to the process of rendering an article or area
T h e process of sterilisation is used in microbiology free ot danger from contam inant, including
for preventing contamination by extraneous microbial, chemical, radioactive and other hazards.
organisms, in surgery tor maintaining asepsis, in The various agents used in sterilisation cun be
food and dmg manufacture tor ensuring safety from classified as follows :
c o n ta m in a tin g organism *, and ill many o ilie r
A . P h y s ic a l agen ts
situations. THe methods or sterilisation employed
1. Sunlight
depend on the purpose for which it is carried out,
2. Drying.
the materia! which has to be sterilised am! the
3. ],Vv hcati flaming, incineration, hot air.
nature of the microorganism* that are to be removed
4. Moi st hea r: pasteurisation, boiling, steam under
or destroyed.
normal pressure, sream under pressure.
Sfctilrsjbon is defined as the process by which
5. nitration: candles, asbestos pads, membranes.
an article, turface or medium is freed of all living
6. Radiation.
microorganisms either in [lie vegetative or spore
state. D^rfrrtionniEUy the destruction or removal 7. Ultrasonic and sonic vibrations.
of all pathogenic organisms, or organisms capable B , C h e m ic a ls
of giving rise to infection. The term Aiitisepsit is 1. Alcohols: ethyl, isopropyl, irichlorobutanol.
used to indicate the prevention of infection, usually 2. Aldehydes: formaldehyde, glutaraldchydc,
by inhibiting the growth of bacteria in wounds ur 3. Dyes.
tissues. Chemical disinfectants which can lie safely 4. Halogens,
applied to sit in or mucous membrane amt are used i. Phenols,
to prevent infection by inhibiting [he growth of 6. Surface-active agents.
bacteria arc called antrstynths. BaC ttiicid^l agents 7. Metallic salts.
or geftnitrides are those which arc able to hill 5. Gases: ethylene oxide, formaldehyde, beta
bacteria. Bacteriostatic agents only prevent the propinlactone.
multiplication ot bacteria which may, however,
remain alive. A chemical which is bactericidal at a
particular concentration, may only be bactericsWiJtic Sunlight possesses appreciable bactericidal activity
at a higher dilutioTl- and plays an important tolc in the spontaneous
Clcaning, though a routine nontechnical sterilisation that incurs under natural conditions.
procedure, plays an important preparatory role The action is primarily due to its content ot

C o p y rig h te d m aterial
 * Ste'i.i-^iion and Disinfection ►
ultraviolet rays* most of which, however, arc
screened out by glass and the presence of ozone in
the outer regions of the atmosphere, Under natural
conditions, its sterilising power varies according to
ci rcum stances. Direct sunlight, as in tropical
countrYcde where il is not filtered off by impurilK's
in the atmosphere, has an active germ hula! effect due
to the combined effect of ultraviolet and heat rays.
Semple and Grieg showed Ui.u in Lidia* typlioid bacilli
exjTosed lo the sun on pieces of while drill doth were
killed in two hours, whereas controls kept In the dark
were still alive after six days- Bacteria suspended in
water are readily destroyed by exposure to sunlight.
I) it 'i i \ t ;
Moisture is esSenlml for the growth of bacteria.
Four-fifths of the weight of the bacterial cell is
due to water. Drying in air has therefore* a
deleterious effect on many bacteria. However, this
method is unreliable and is only of theoretical
interest. Spores are unaffected by drying.
I l l - \T
Heat i-; the most reliable method of sterilisation
and should be the method of choice unless
contraindicated. Materials char may be damaged
hy heat can be steri li-erl at lower temperatures, for
longer periods or hy repeated cycles. The factors
influencing sterilisation by heat arc:
1- nature of beat— dty heat or moist heat,
2. temperature and time,
1 number of micToorgun isrtis present*
4 . character^ I ic s o f the organisms* such as, species*
strain* sparing capacity, and
5, type of material from which the organisms have
to be eradicated.
T h e killin g effect o f dry heat is due to protein
dvnatuT.ition, oxidative J,image and Lhe toxic effect
of elevated levels of electrolytes. T h e lethal effect
of moist heat is due to the denaturation and
coagulation of protein. The advantage of steam lies
in the latent heat Eiherated when it condenses on a
cooler surface, raising the temperature of that
25
surface, In the case of the spore, steam condenses
on it, increasing :ts water content with ultimate
hydrolysis and breakdown of the bacterial protein.
In a completely moi-ture-free atmosphere, bacteria,
like many proteins, are more reaislant to heat. They
are killed when oxidation of the cell constituents
occurs and this requires much higher temperatures
than that needed for coagulation of proteins.
The time required for sterilisation is inversely
pToportiunaE to the temperature of exposure and
can be expressed as "thermal death time', which is
the in-:nimum time required to kill a suspension of
organisms it a predetermined temperature in a
spec:lied environment. The sterilisation time is
related to the number of organisms in the
suspension, presence or absence of spores, arid the
strain and characteristics of organism. The
recommended minimum sterilising nmes do not
include the lime taken to reach the specified
temperature. 'I*he nature of the material in which
the utjmi 1: is are heated affects the rate of killing.
The presence of organic substances, proteins, nucleic
acids, starch, gelatin, sugar, fats and oils, increase
the thermal death time. The presence of
disinfectants and high acid or alkaline pH hasten
bacterial killing.
I >HY H E A T
F la m in g Inoculating loop or wire, the tip of
forceps and searing spatulas are held in a bunsen
flame till they become red hot. Inoculation loops
carrying infective material may be dipped in a
disinfectant before flaming to prevent spattering.
Incineration: This is an excellent method for
safely destroying materials such as contaminated
clorh, animal carcasses, and pathological materids.
Plastics such as PVC and polythene can be dealt
with similarly but polystyrene materials emit clouds
of dense black smoke and hence should be
autoclaved in appropriate containers.
H o t uir oven: This is the most widely used
method of srerills, ilion by dry heat, A luoMing period
of 160 "C for one hour is used to sterilise glassware*
C o p y rig h te d m aterial

forceps, scissors, scalpels, all-glass syringes, swabs,
some pharmaceutical products such as liquid
paraffin, dusting powder,, fils and grease Hot air lS
a had conductor of heat and its penetrating power
is low. The oven js Usually li cured hv electricity
with heating elements in the wall of the chamber.
11 must be fitted with a fin to ensure even
distribution (]f air and elimination of air pockets
(Fig. 3.1). It should not be overloaded. The material
should be arranged so as to allow free circulation
of air in between the objects. Glassware should be
perfectly dry before being placed m the oven. Test
tubes and flask* should be wrapped in paper.
Kubbcr materials, except silicon rubber, will not
withstand the temperature. Ac 160 LC cotton plugs
may get chaired. Cutting instruments such as chose
Used in Ophthalmic surgerv, should ideally be
sterilised for two hours at t SO ^C. The British
Pharmacopoeia recommends a holding time ot one
hour at 150 for uils, glvverul and dusting
powder. The oven must be allowed lo cool slowly
for about two hours before the door is opened, since
the glassware mar crack due to sudden or uneven
The sports of a
nontoxigenic strain of CJaftriditEun telmr are used
as a microbiological test of dry heat efficiency. P&pcr
strips impregnated with 101' spores are placed in
envelopes and Inserted into suitable packs. After
sterilisation, the strips are removed and inoculated
info t hiejglycol Ia te or cooked meat media and
incubated for sterility test under strict anaerobic
conditions for five days ac 37 'C
A Brownes tube (green spot) is available for
dry bent and is convenient for routine use. After
proper sterilisation a green colour is produced (after
6U minutes at 160 DC or 115 minutes at 150 cC).
Therm ocouples may also be used periodically.
The milk is
heated at either 63 “t for 30 minutes (the holder
method) or 72 QC for 15-20 seconds (the flash
process) followed by cooling quickly to 13 °C or
lower. By these processes all uonsporiiig patliogens
such as mycobacteria, bruccllae and salmonellae
are destroyed. C o x i d l j h u t n e t i i is relatively beat
resistant and mav survive the holder method.
V a c c in e s o f nt 2 n f p o rin g b a c te ria are heat
inactivated in special vaccine barbs at 60 ° C for
o n e h o ur. S e t utn o r b o d y flu id s containing
cnagulahlc proteins can he sterilized hv heating for
one h o ur at 0 6 cC in a w ater bath on several
successive days.
Media auclt as Lowcnstcio—Jensen and
Locrtlcrrs scrum arc rendered sterile by heating at
HO-^5 “C lor half an hour ort three successive duvs
in an jnspj.ssaCor.
Though practically all mesnphilic nonspnring
bacteria are killed by exposure to moist heat at 60 °C
for 3 0 minutes, Staphylococcus aureun and
5 frcprovoccrcus faecalis require 60 minutes. A
temperature of SO QC for 5 -1 0 minutes destroys
the vegetative forms of all bacteria, yeasts and
moulds. Among the most heat resistant cells arc
the spores of C i o t t r i d i u m b o t u i i n u m which require
C o p y rig h te d m aterial
120 Kl for four minutes, or 100 for 330 minutes
for their destruction. .Most viruses ire inactivated
very rapidly it 60 “C , bm some are relatively
resistsitt, for example, poliovirus and hepatitis U
virus which may survive for 30 minutes and over
10 hours respectively it this temperature.

Vegetative bacteria ate killed almost

ini mediately at 9Q-10Q °C, but sporing bacte ria
require prolonged periods of boiling. Boiling ls nor
recommended 1l>: sterilising of instruments used
for surgical procedures and should he regarded only
a$ i means of disinfection. Nothing shore of
autoclaving at high pressure can destroy spores and
ensure sterilisation, Hurd water should not be used.
Sterilisation mav be promoted by the addition of
2% sodium bicarbonate to the water.
In cases where boiling is considered adequate,
the material should be immersed in the water and
boiled for 10-30 minutes. The lid of the steriliser
should not be opened during the period.
An atmosphere of free steam is used to
steriliM culture media which may decompose if
subjected to higher temperatures. A Koch or Arnold
steamer is usually used. (Laboratory autoclaves can
also be used for this purpose-; This is an
inexpensive method.llic container and medium are
simultaneously sterilised, evaporation from the
medium Is prevented and the apparatus requires
little or no attention.
The steamer consists of a tinned cupper cabinet
with the walls suitably lagged. The lid is conical,
enabling drainage of condensed steam, and a
perforated tray fitted above the water level ensures
that the material placed on it is sti mounded by steam
(Fig, 3 J ) . A single exposure of ninety minutes
usually ensures sterilisation hut for media COOraining
sugars or gelatin an exposure of 100 **C for 20
minutes un three successive days is used. This iS
known as tv/nJaJJrvarj'ojl oi in ferrUrfter]f >[cri/jy j(j□ j j .
The principle is [bat the iirst exposure kills all
vegetative bacteria, and the spores, since they are
in a iavourihle medium, will germinate and he killed
on the subsequent occasions. Though generally
adequate, this method may fail with spores ot
certain anaerobes and therm ophilcs.
The principle of the
autoclave or steam steriliser is that water boils when
its vapour pressure equals that ot the surrounding
atmosphere. Hence when pressure inside i closed
vessel increases, the temperature at which water
boils also increases. Saturated steam has penetrative
power. When steam comes into contact with a
c<wder surtace it condenses to water ami gives up
irs latent heat ro that surface (lbOO ml steam at
lOQ’X-’ and at atmospheric pressure coruien$£s into
one ml of water at 100 and releases 518 calories
of heat). The large reduction in volume sucks in
more steam to the area and the process continues
rill the temperature of that surface is raised to that
of rhe steam. The condensed water ensures moist
conditions lor killing the microbes present.
Sterilisation by steam under pressure is carried
out at temperatures l>etween 10K UC and f47 ^C.
By using the appropriate temperature and time, a

C o p y rig h te d m aterial
variety of materials such as dressings instmmerits.
Laboratory ware, media a]id pharmaceutical
products can he sterilised- Aqueous solutions arc
sterilised between IQS *C and 116 VC, Heat is
conducted through the walk of the sealed
containers until the temperature of the fluid inside
is the same as that of the steam outside.
Several types of steam sterilisers are in use:
1. laboratory autoclaves,
2. hospital dressing sterilisers,
3. bowl and instrument sterilisers, and
4 . rip id cooling steriLisers-
Even the domestic pressure cooker can be used
as a steriliser.
In its simplest form, the laboratory autoclave
consists of avertical or harfaincal cyimder ofguiunetal
or stainless Steel, in ,|.Supporting sheet iron case. The
lid or dour is listened by screw clamps ami nude
airtight by a suitable washer The autoclave has on its
lid or upper side a dischaigc Tap for iir and steam, a tend to boil violently and spill from rhe container
pressure gauge wid a safety valve that tan be set to and sometimes an explosion may occur. If opened
blow off at any desired pressure. Heating is. by gas or after the pressure inside h i- fallen below
electricity (Fig. 3.5). atmospheric pressure, an excessive amount of water
Sufficient water is pur in rhe cylinder, the would have evaporated And lost from the media.
material ro be sterilised is placed on the tray, and The defects in this type of autoclave arc:
the autoclave is heated. The lid is screwed tii^ht 1. hHic method of ajt discharge is inefficient, and
with the discharge tap open. The safety valve is it is difficult to decide wlnn the discharge is
adjusted to the rec|Uired pressure. The steam-air complete. It the ait is nor completed removed,
mixture is allowed to escape freclv rill all the ait the desired temperature wiU not be attained.
has been displaced- Thai cun be tested by leading 2r There is no facility for drying the toad after
the escaping steam into a pail of water through a sterilisation and before taking it out.
rubber tubing. When no more air bubbles come The domestic pressure cooker serves as a
out in the pail the discharge tap is closed. The miniiture autoclave and maybe used for sterilising
steam pressure rises inside and when it reaches the small articles in clinics and similar estabBuhtfrentx.
desired set level, the SAtcrv value opens and the A wide variety of au toe lives have been
excess steam escapes. From this point, the holding unnufaLturcd incorporating various devices for
period is calculated. When the holding period is overcoming these defects and other difficulties in
over, the heater is turned off and the autoclave workj ng.
allowed to coot till the pieHsure gauge indicates For determining die
chut the pressure inside is equal to the atmospheric efficacy of m<11-r hear btr ri Ii^^.ti- >a, sprires ■)f H.idHus
pressure. The discharge tap is opened slowly and ftcm thcrm ophilus are used as the rosi organism*
air is let into the autoclave. If rhe tip ls opened I his is a thermophiEc organism with in optimum
when the pressure inside lS high, liquid media will growth temperature of 55-60 C and its spores

C o p y rig h te d m aterial
require an exposure of 12 minutes at 121 "'C to be
kilBedi. Paper strips jmprcgnated with If)6 spores
are dried at room temperature a ltd placed in paper
envelopes, These envelopes are inserted in different
parrs o f tile load. After Sterilisation, the strips are
inoculated into a suitable recovering medium and
incubated for sterility test »t 55 aC for five days.
Chemical indicators, autoclave rapes and
thermocouples may also be used instead.
Filtration helps to remove bacteria from heat labile
liquids such as sera and volutions of sugars or
antibiotics used for preparation of culture media-
As viruses pass through ordinary filters, filtration
can be used to obtain bactcria-fiee filtrates of clinical
samples for virus isolation. Fiber discs ilto help to
concentrate bacteria from liquids as for example in
testing water samples for cholera vibrios or typhoid
bacilli. Bacterial toxins can he obtained by passing
cultures through filters.
T h e following types o f filters have been used.
T h e s e are m an u factu red in
d rile rent grades of porosity ami have bee-n used
widely for purification o f water for industrial and
drinking purposes. They are o f two types: (a) U n-
gl.i/L'd ceramic fillers (for example, Charnherbud
and Doullon filter; (b) Diatuinaceou-s earth filters
(for example, Berkefold and Mandler filters).
are disposable, single-use discs.
They have high adsorbing capacity and tend to
alkalinisc filtered liquids. The carcinogenic
potential of asbestos has discouraged their use.
EvampUs aw Seit?- and Stcritm! filter;.
arc prepared by hcat-
fu&ing finely powdered glass panicles ofgraded sizes,
Thev have Low absorptive property and can he
cleaned easily bur are brittle and expensive,
made of cellulose esters or
other polymers have largely replaced other types
of filters., They are routinely used in water
purification and analysis, sterilisation and sterility'
testing, and for the preparation of solutions for
parenteral use, They come in a wide range of average
pore diameters (APD), rhe 0.22 mm size being
most widely Lised for sterilisation.
Two types of radiation arc used for sterilisation,
non Ionising ,md ionising. Infrared and ultraviolet
rays are of die nonionising low energy type, while
gamma rays and high energy electrons are the high
energy' ionising type.
. Here electromagnetic
rays w ith wavelengths longer than those of visible
Light are used. These are to a targe extent absorbed
as heat- l lence infrared radiation can he considered
as a form of hot air sterilisation, Infrared radiation
is used for rapid mass sterilisation of prepacked
items such as syringes and catheters. Ultraviolet
radiation is used for disinfecting enclosed area? such
■ls entrywavs, operation theatres a n d laboratories.
X-rays* gamma rays and
CO&mit ray? are highly lethal to l)NA and other
C o p y rig h te d m aterial
viral constituents. They have very high penetrative
power. Since there is no appreciable increase in
temperature in this method, it is referred lo as cold
sterilisation, Commercial plants use gamma
radiation for sterilising ireim like plashes, syringes,
swabs, catheters, animal feeds, cardboard, oils,
greases, fabrics and metal toils.
I Ugh energy electron radiation as a method of
sterilisation is not widely used in medicine.
Ultrasonic and sonic waves arc eredired with
bactericidal powers hut the results have been
variable. Microorganisms vary in their sensitivity
to diem and survivors have been found alter such
treatment. Hence this method is of no practical
value in sterilisation and disinfection.
Several chemical agents are used as anti optics and
disinfectants. However little is known about the
mechanism of action of many of these agents. An
ideal antiseptic or disinfectant should :
■ have ,l wide spectrum of activity and must be
effective against all microorganisms, that is,
bacteria including spores, viruses, protozoa and
fongi;
* be active in the presence of organic matter;
■ be effective in acid as well ,ls alkaline media;
* have speedy action;
* have high fjenetraung }H)wcri
* he stable;
* be compatible with other antiseptics and
disinfectants;
* not cornicle metals;
* not cause local irritation or sensitisation;
* not interfere with healing;
* not be toxic if absorbed into circulation;
* be cheap and easily available; and he safe and
easy to use.
Sucll an ideal chemical is yet to l>e found.
The factors that determine the potency of
disinfectants arc:
* cn mien [ration of die substance;
* time of action;
■ p H o f the medium;
* temperature;
* nature of the organisms; and
1 presence of extraneous material;
Chemical agents act in various ways. The main
modes of action are;
1. protein coagulation;
2, disruption of cell membrane resulting in
cvpo:-ure, damage nr lo>s of the Contents;
3, removal of free sulphydiyt groups essential for
lJl£ fnnci toiling of the enzymes; and
4. vubutrace competition: A compound resembling
rhe essential substrate of the enzyme diverts or
misleads the enzymes necessary for the
metabolism of the cell and causes cell death,
Ethyl alcohol (ethanol} and isopropyl aleuliol are
the most frrtjutntiv used. Tbev art used mainly as
skin antiseptics and act by denaturing bacterial
proteins. They hive no action on spores. To be
effective, they must be used at a concentration of
6(b-90 per cent in water. Protein slows its action
whereas 1% mineral acid or alkali enhances the
action. Isopropyl llculioi is pie Ierred as it is a better
fat solvent, mote bactericidal and less volatile. It ls
used for rhe disinfection of clinical thermometer;.
Methyl alcohol is effective against fungal spores
and is used for treating cabinets and incubators
affected by them. The insides of the chambers arc
wiped with liberal amounts of methanol. A pad
moistened with methanol and a dish tii water (to
ensure high humidity) ate kept inside, and rhe
incubator is left at working temperature for several
hours. Methyl alcohol vapour is toxic and
inflammable.
Formalclchwlc is active against rhe amin-o group in
the protein molecule. In aqueous solutions, it is
markedly bactericidal and sporiddal and also has a
C o p y rig h te d m aterial
lethal effect on viruses, [t is used to preserve
anatomical specimens, am! far destroying umbra*
sjKtres in half and wooh 10% IbnriaUn containing
0.5% sodium tetraborate is used to sterilise clean
metal instruments.
Formaldehyde gas is used for sterilising
instruments and heat sensitive catheters and for
fumigating wards, sick rooms and laboratories.
Under properly controlled conditions, clothing,
bedding, furniture and books can be satisfactorily
disinfected.
The gas is irritant and toxic when inhaled.
Surfaces which have been disinfected by this agent
■nay give off an im [.lot vapour for some time after
disinfection. IT is can he nullified by exposure to
ammonia vapour Lifter disinfection hai been
completed.
This has an action similar to
formaldehyde. 11 is specially effective against
tubercle bacilli, tungi and viruses. It is less toxic
and irritant Co the eves and. skin titan formaldehyde.
It has no deleterious effect on the cement or tenses
of instruments such as cystoscopes and
hr one ho scopes, ll can be Safely used to treat
Corrugated rubber anesthetic Lubes and lace mask:-,
plastic endotracheal tubes, metal instruments and
polythene tubing.
Two groups of dyes, aniline dyes and acridine dyes
are used extensively as skin and wound antiseptics.
Both arc haetcriostatic in high tlilution but arc of
low bactericidal activity, 'fhc aniline dyes in use
are brilliant green, malarhireguften and crystal viofer.
They ire more active again sit Gram positive
organisms than against Gram negative organisms,
They have no activity against tubercle bacilli, and
hence the use of malachite green in the
Lowenstcin-Jen sen medium. Though they are
nonirritant to the tissues and nonius in, they sue
considerably inhibited hy organic material such as
pus. Their lethal effects on bacteria are believed On
be due to their reaction with the acid groups in the
cell. These dyes arc used in the microbiology
laboratory as selective agents in culture media.
The acridine dyes arc mote active against Gram
positive organisms than against Cram negative but
are not as selectve as the amline dyes. They are
affected very little by the presence of organic matter.
The more important dyes are proflavine, acriflavinc,
mtlavinc and aminacrine. They show no significant
differences m potency,! f impregnated in gauze, they
are slowly released in a moist environment, and
hence their advantage and use in clinical medicine.
They impair the DNA complexes of the organisms
and thus kill or destroy the reproductive capacity
of the ceJL
Iodine in aqueous and alcoholic solution has been
used widely as a skin disinfectant. It is actively
bactericidal, with moderate action against spLire-s.
it is active against the tubercle bacteria and viruses.
Compounds of iodine with nonitmic Wetting of
surface active agents known as iodophores are
claimed to be more active than the aqueous o i
alcoholic solutionis of iodine.
Chlorine and its compound? have been used as
disinfectants for many years. Water supplies,
swimming pools, food and dairy industries use
chlorine for disinfection. Chlorine is used
com mooli1 as hypochlorites. Chlorine .1 ltd
hypochlorites arc markedly bactericidal. They have
a wide spectrum of action against viruses. The
organic chloramines are used as antiseptics tor
dressing wounds.
These are obtained by distillation of coal tar
between temperatures of 1 7 0 ^ and 2 7 0 “C, Lister,
the father of antiseptic surgery, first introduced their
use in surgery { 136 ?). Since then a wide range of
phenolic compounds have been developed as
disinfectants. The lethal effect of phenols is due to
their capacity to cause cell membrane damage,
releasing cell contents and causing lysis. Low
C o p y rig h te d m aterial
co n cen tratio n s o f p heno l fire d e r a t e proteins.
M em brane-bound oxidase* and dehydrogenases arc
ir activated by concentrations o f phenol that are
rapidly bactericidal for microbes.
Phenol (c a rb o lic a c id ) is a p o w e rfu l
m icrobicidal substance. T h is and other phenolic
disinfectants derived from coal car me widely used
as disinfectants for various purposes in hospitals,
Lyso! and crcsol are actinic against a wide range of
organisms. T h e y are not readily inactivated by [Ire
presence o f organic matter and are thus good general
disinfectants. T h e se are toxic to humans. Various
proprietary preparations or form ulations ot phenol
■3re in w ide use. T h e related chlorophenols nnd
cUorOKYphenols, [hough less toxic and irritant, un­
less active and more readily inactivated by organic
matter. Hoth these gre>ups o f substances arc relatively
in a c tiv e a g a in s t P se u d o m o n a s . V a rio u s
com hinations of these are used in the control o f
pyogenic cocci in surgical and neonatal units in
h o spitals.I IcxachDorophene is potentially toxic and
should be used with. care. Chlo rhexidine ( H i hit arc)
in a relatively no ntenk skin antiseptic most active
against Gram positive organisms and fairly clfeciive
against G ra m negative onei. Aqueous solutions are
used iti the treatment o f wounds.
T h is ir a colour lens liquid with
a boiling point o f 1 0.7 and at m irma] temperature
and pressure is a highly penetrating gas with a sweet
ethereal sm ell. It is h ig h ly inflam m able and in
co n cen tratio n s in a ir greater than 3 % f h ig h ly
explosive. Ely m ixing it w ith inert gusts such is
carbon d boride or nitrogen, to a concentration o f
] 0 % , its explosive tendency is eliminated.
Its action is due to its alkylating the am ino,
carboxyl, hydroxyl and sulphydryl groups in protein
molecules. In addition, it react* w ith [3 N A and
ft N A , Its use as a disinfectant preseni:- a potential
toxicity rn human beings, including mutagenicity
and carcinogenicity, It is effective against oil tvpcs
o f microorganisms including viruses and spores,
It d iffu se s rhrnugb m a n y types o f porous
materials and readily penetrates some plastics. It is
SpeCLaUv used for Sterilising lie art— lung machines-,
respirators, sutures, dental equipment* books and
clothing. It is unsuitable tor fum igating room s
because ot its explosive property. It has. been
su cce ssfu lly used to sterilise a w id e range o f
materials such as glass, meta] and paper surfaces,
clothing, plastics, soil, some foods and tobacco. It
is Lintailt, and personnel working with it have to
take strict precautions.
T h is is w idely em ployed
tor fum igatio n o f operation theatres and other
rooms. After sealing the windows and other outlets,
formaldehyde gas is generated hy adding 15 0 g o f
K M n O , to 2EG ml form alin for every 10 0 0 cu. ft
(.2$.3 CU. m) o f room volume. T h e reaction produces
considerable heat, and so heat resistant vessels
■ should be used. A f t e r s ta rtin g ge n eratio n o f
formaldehyde vapour, the doors should be scaled
and left unO]»eiled. for h<iurs.
T h is is a
condensation product o f ketane and formaldehyde
with a boiling point o f 16 3 *C, T h o u g h as a gas it
has low penetrating power, it is said to be more
efficient tor fumigating purposes than formaldehyde.
It has a rapid biocidal action but unfortunately has
Carcinogenic activity. Fo r sterilisation o f biological
products 0 .2 % B P L is used, lr is capable o fk illin g
all microorganisms and is very active against viruses.
S ubstances w h ich alter energy re la tio n sh ip at
interface h, produeing n reduction o f surface or
interfhcial tension nrc referred to as surface active
agents- T h e y are w idely used as w etting agents,
detergents and emulsifiers. T h e v are classified into
four m idii groups, anionic, cationic, nottionic and
am p h o teric. T h e most im po rtant antib acterial
agents are the cationic surface active agents. These
act on the phosphate groups o f the cell membrane
and also enter the c e ll T h e m em brane loses its
sc m i p e r m e a b ility an d d ie c e ll p ro te in s are
C o p y rig h te d m aterial
 * SlerilisaliQP and Disinfection > 33

denatured. The cationic compounds in the form of
quaternary ammonium compounds are markedly
bactericidal, being active against Gram positive
organisms and ro a lesser extent on Gram negative
ones. They haw no action on spores, tubercle bacilli
and most viruses. The common compounds arc
acetyl tri methyl ammonium bromide (Cetavion or
cetrimide) and benzalfconiuin chloride. These hit
most active at alkaline pH. Acid inactivates them.
Organic matter reduces their action and anionic
surface active agents., like Otdinarv soaps, render
them inactive. The anionic compounds, e.g,,
common soap, have moderate action- Soaps
prepared from saturated fatty acids (such as coconut
oil) are more effective against Gram negative bacilli
while those prepared from unsaturated fatty acids
(oleic acid) have greater action against Gram
posinvc and Neisseria group of organisms. The
amphoteric or ampholytlc compounds, known a&
' J l'uo' compounds, are active agiiinst 9 wide range
of Gram positive and Gram negative organisms and
some V:Tuses. These, however, are non in general
use.
M e t a l l ic S alts
Though all salts have accrtnin amount of germicidal
action depending on their concentration, salts of
heavy metals have a greater action- The salts of
si Iwr, copper and mercury are used as disinfectants.
They are protein Coagulants and have the capacity
to combine with free sulphydryl groups of cell
enzymes, when used at appropriate concentrations.
Mercuric chloride, once used as a disinfectant is
highly toxic. The organic compounds, thbmercal,
phenyl mercury nitrate and mercurochrome, are less
toxic and are used as mild antiseptics and haw a
marked bacieriostsLtic., hut a weak bactericidal and
limited fungicidal action. Silver salts in aqueous
solution have a limited use. C o p p er salts arc used
as fungicides.
T e s t in g o i■ D is in f e c t a n t s
flic re is no single reliable test available to
determine efficiency of a disinfectant. This is due
to the number of parameters which influence
disinfectant activity. Traditionally In such tests
phenol is taken as the standard- In the Ridcal
Walker test, vuspensinns containing o^ual numbers
of typhoid bacilli are submitted to the action of
varying concentrations of phenol and of the
disinfectant to be tested. The dilution of the rest
disinfectant winch sterilises the suspension in a
giwn lime, divided by the correspond!ng dilution
of phenol is stated as the phenol coefficient (Phenol
= 1) of the disinfectant. This test does not reflect
natural conditions as the bacteria and the
disinfectant react directly without any organic
matter being present. Modifications have therefore
been suggested- In the Chick Martin test, the
disinfectant acts in the presence of organic matter
(dried ye&ST dr feces). Even this modification falls
short of si mula ri r.g natural conditions. Various other
modifications have been introduced, but no test is
entirely saiisfactory.

Further Reading
Block 55 i■:.!'■ 1991. Chemical Disinfection in Hospitals, 2 edn. Loudon, Public Health Laboratory Service.
Gardner JK- and MM- Ptd 1991. Jrrmxkuticifl JUStcfiUntion nnd DifinfccTiont£ *d tL . Edinburgh; Churchill Livinptonc.
RusMill AD er a]. 1992. Principle* tnd Ptutiiix ol'iJijrii.fttri rtn, S(en!r\itfoo nnd Piwtvji tii 2'^ cd-n. Oxford: Blackwell
5i irntific.

C o p y rig h te d m aterial
 Culture Media
Bacteria have to t>c grown (ctdhiredj fur them to
be identified, us oply rarely can they he recognised
hy their morphology atone. 'H it *mdy o f bacteria
involves the study of bacterial populations rather
than o f single bacterial cl'II h. In the hum an and
animal bodies, as well as in other natural scurnpen,
bacteria occurs* mixed populations. By appropriate
procedures they have to hc grown separately
(isofoted} tm cifJrunf media and obtained as pure
cultures lor study.
Numerous culture media have been devised.The
original media used by Louis Pasteur were liquids
such as urine or meat broth. Liquid media have
many disadvantages. Bacteria ^rm nii; in Liquid
media may not exhibit specific characteristics for
their identification. It is also difficult to isolate
different tyyies ol bacteria Jrum mixed population?,
using liquid media. However, 1i<|uilI media have
their uses, for example, tur obtaining bacterial
growth from blood or water when large volumes
have to be tested, and for preparing hulk cultures
of antigens or vaccines.
W h ile bacteria giW v diffusely in liquids, they
produce discrete visible growth on solid media. 11
in o cu late d in suitable d ilu tio n s, bacteria form
coJonies, w hich are clones of cells originating from
a single bacterial cell. O n solid media, bacteria have
distinct colony morpholt igy and exhibit many other
ch aracte ristic features such as pigm entation or
hemolysis, m aking identification easy, 'flic earliest
solid m eJ i uni was rooked , i Lt pntati > be If (ibert
K o ch. Later he introduced gelatin to solidify liquid
m edia but it was pot jiatis factory as gelatin is
liquetied at 24 "C anti also by insirn proteolytic
bacteria. The use of agar to solidify culture media
was suggested bv Trau Hesse, the wite ol one o f
the investigators in Koch’s laboratory, who bad seen
her mother using agsu for making Jellies!
Agar [or agar-agar) is now universally used
for preparing solid media. Agar is obtained from
some types uf seaweeds. Its chief constituent is a
king chain polysaeehuidt, It also contuins varying
amounts of inorganic salts and small quantities of a
protein- like substance. It ha* virtually no nutritive
value and is not affected by the growth of bacteria.
Agar is hydrolysed at high temperatures* at high
acid or alkaline pi I. 1^ unique property is that it
melts lil -’C and usually sets tit 42 "C depending
on agar eoncentraiion Approximately 2% agar is
employed tor solid media. The jellitying property
varies in different brands of ogpr; for example, New
Zealand agar bus more jellifying capacity thaul
Japanese agar. Agar is manufactured either in long
shreds nr as powder. There may be variations
briw ern different hutches o f the same brand, apart
from rhi.- differences due to a different source of
agar.
Another almost universal ingredient of common
media is jjeplune. It is a complex mixture o f partially
digested proteins. Its constituents arc proteoses,
jjtilyjtepiides and anunnaods* :lvariety of inorganic
sabs including phosphates, potassium and
nugnerium and certain icceHory growth factors
such as nbiaflaviit. D iiFercnl b u n d s of peptone show
appreciable differences in composirion and growth
promoting properties, fherc may he variations
between different batches of the same brand,
''fiecial brand? of peptone such as neopeptone
C o p y rig h te d m aterial
 ■ Cuhune M edia *■ 35

vlridjin streptococci of £pfttheria tecMI

C o p y rig h te d m aterial
M 4 lexttwofc d MieroWotogy ►

C o p y rig h te d m a la ria l
and pntconc peptone arc available for special use-:.
Commercially available peptones or digest brotSi dm
he used- Meat extract Ls also available CornrncfCuiDy
and is known as I >ah-Lcm«). Blood, scrum and yeast
extract arc other common ingredients.
Media have been classified in many wap:
1. Solid media, liquid media, scmisolid media.
2- Simple media, complex media, synthetic or
defined media, semidefined media, speck!
media, Special media are further divisible into:
enriched media, enrichment media, selective
media, indicator or differential media, sugar
media, and transport media.
3. Aerobic media, anaerobic media.
An example
is nutrient broth. 1[ consists of pep; one, meat Extract,
sodium chloride anti water. Nutrient agar, made: by
adding 2% agar to nutrient broth is the simplest
ami most common medium in routine diagnostic
laboratories. If the concentration oi ,ig:)i is reduced
to 0,2-0,5% , semisolid or sloppy agar is obtained
which enables motile organisms to spread. Increasing
the concentration of agar to prevents spreading or
swanning by organisms such as Pfotcus.
T lic s c have atlded ingredients
for special purposes or for bringing o«t certain
characteristic-; or providing special nutrients required
for the growth of the bacterium under study.
These media
arc prepared from pure chemical substances and
the exact composition of the medium Is known.
These arc used for various special studies such as
metabolic requirements. Simple peptone Water
medium, 1% peptone with 0,5% NaCl in water,
may be considered a scmidcfmed medium since its
composition is approximately known.
In these media, substances such
as hhxid, serum, or egg are added hi a hasal medium.
They sue used to grow bacteria which are more
exacting in their nutritional needs. Examples arc blood
Ligur, chuCaljte ;igUr and egg iftediA.
In mixed cultures or in
materials containing more chan one bacterium, the
bacterium to be Isolated is often overgrown hy the
unwanted bacteria. Usually the nonpathogenie or
commensal bacteria tend to overgrow rhe
pathogenic ones* tor example S.typhi being
overgrown bv E. coii in cultures from t’eccs. In such
situations, substances which have a stimulating
effect on the bacteria to he grown or an inhibitory
effect on those ro be suppressed am incorporated
in rhe medium. If such substances are added to a
liquid medium* tlic result is an absolute increase in
the numbers: of the warned bacterium relative to
rhe other hacteriiL. fiuch media are called
enrichment media, for example lelrathionate broth
where fbc rctmthionatic inhibits coli forms while
allowing typhoid-paratyphoid bacilli to grow freely,
and Selim itc ]■' broth for dysentery bacilli.
As in the above case, if the
inhibiting substance is added to a solid medium, it
enables a greater number of the requited bacterium
to form colonies than the other bacteria, for example,
dcsoxychobre citrate medium for dysentery bacilli.
Such solid media are known as selective media.
rbese m edia con tail) an
indicator which changes colour when a bacterium
grows in them, foe example incorporation of sulphite
in Wilson and blsir medium, S, typhi reduces
sulphite to sulphide in the presence <if glucose and
the Colonies <xf 5 . typhi have a black metaJlic sheen.
Potassium tellurite lei McLeod's medium is reduced
to metallic tellurium hy the diphtheria bacillus To
produce black colonies.
A medium which has
substance incorporated in it, cnahling it to bring
out differing charactcrisi-foii of bacteria arul. thus
helping to distinguish between them* is called a
differential medium. For example, MaeConkey’s
medium which consists of peptone, lactose, agar,
neutral red and taurocholaic shows up lactose
fermenters as pink colonics, while non lactose
fermenters ire colourless or pale. This may also be
termed indicator medium.
C o p y rig h te d m aterial
IE i Textbook of Microbiology *

Some of These terms for media arc
interchangeable. For example, the blood agar
medium i&10 enriched medium but bactena ly-mg
red ceils show a clearing around their colories.
Thus, d in an indicator medium .m -well. There are
many special mrdi.L lot dem onstrating particular
character i-tics, [ike Nader's medium which enables
us to view lee ith iri.i-.L- activity.
Su gar media: 'the term 'sugar In microbiology
denotes any fermentable substance. They may be:
1. Monosaccharides —a) pentoses, e.g., arahinose,
xylose, b] hcxoscs, e.g,, dextrose, mannose
2. Disaccharides, e.g., saccharose, lactose
3- FblyMCL-hariilcs, e.g., starch, tnulin
4. Trisaceharides, e.g., raffinose
5. Alcohols, c,g„ glycerol, sorbitol
6. Gluco&tdes, e.g., saliein, aesculin
7. Noncarbohydrate substances, e.g., inositol.
The usual sugar media cons ist of 1% of the sugar
in peptone water along with an appropriate
indicator. A small tube (Durham's tube) is kept
inverted in the sugar tube to detect gas production.
For organisms which arc exacting in their growth
requirements (for example pneumococcil1, Hiss’
serum sugars arc used. They contain 3% serum.
T ra n s p o r t m ed ia: In the case of dd-cate
organisms (like gonococci) which may not survive
the time taken for transporlng the specimen to the
laboratory or may be overgrown by nonpathogens
(such as dysentery or cholera organisms in fcccs),
special media are devised for transporting the
specimens. These are termed transport media, for
example, Stuart 'smedium— a non~nutrient soft agar
gel cont.i ini: ig 1 reduc ing agent to prevent oxidation,
and charcoal to neutralise certain bacterial
lnhiin.tors— for gonococci, and buffered glycerol
5.iline: for enteric bacilli.
A n a e ro b ic m edia: These media arc used to
grow anaerobic organisms, for example, Robertson's
cooked meat medium.
Various media to test special properties like
urease production, and composite media for
simultaneous demonstration of different features
have been devised. They arc dealt with in the
appropriate chapters.
For identifying prepared media, a colour cude
is usually adopted. This depends on the laboratory'
or group of laboratories. One colour or a mixture
of colours is used on the cotton stopper, or colour
paints are used on the caps.
Culture media used to be prepared in
laboratories themselves, starting with basic
ingredients. Not only was this laborious but it also
led to considerable batch varLiTvm m the quality of
Tnedin, With the teady avail lability of commerci.iJ
dehydrated culture media, the process of media
makuig has become simpler and its quality more
uniform.

Further Rending
Atlaa PM l.C ftfks 199*3. \1ii.Tvbrab%k*} Modi*. Maryland: CRC Press,
CnTWiant R *1 al, 1979, MnJrcal MiuvbJchgY Vol, II, TSt Practice of Medical Mscrov.oiogyt 12"' edn. Londun:
'L h11j r■.-hii11I.ivinptHitlt
Difat Afjrtuaf tti Dth^fwat Culture Mcd'i am/ Pej^nO: 1977. 9thcdn. Michigan: Difco Laboratories.
Oxoid Manual 1976.3rd edn. Basingstoke: Oinid Ltd.
Murray PR et a] 1999. Manual of Clinical Microbiology. 7* edn. Wasliingron DC: ASM Press.

C o p y rig h te d m aterial
 Culture Methods
C ulture m ethods employed depend on the purpose
fo rw h ic h they ire intended. In the clinical laboratory,
the indications for culture are m ainly to:
1. i-solufc bacteria in pure culture"
2, dem onstrate th eir properties;
3- o b ta in s u ffic ie n t g ro w th fo r p re p n m io n of
antigens and for o th er tests;
4. type isolate* by m ethods such ilh bacteriophage
and hactcriorin susceptibility;
5. d e te n u ine sensitivity to snribiotics;
6. estim ate viable counts; and
7. maintain stock Cultures.
Fhe m ethtjds o f culture used ordin arily In the
laboratory are the streak, law n, stroke, stab, p s u r
plate and liq u id cu ltu res. S pecial m e th o d s are
em ployed fo r culturing anaerobic bacteria.
T h e s o a k o j/ftfze (surface p latin g } m etho d is
ro u tin e ly em ployed for the isolation o f bacteria in
pure culture tram conical s]?ecinn£nfiL A ]>LaiiiHim
loop is charged w ith the specim en to be cultured.
O w in g to th e h ig h cost o f p la tin u m * loops fo r
ro u tin e w o rk are m ade o f N ic h ro n tc resistance w ire
{24 S W G size). O n e lo o p fu l o f the specim en is
transferred onto the surface o f a w ell dried plate,
o n w h ic h it is spread o v e r a s m a ll area a t th e
periphery, T h e inoculum is then distributed th in ly
over the plate by streaking it w ith the loop in a
series o f parallel lines, in d iffe re n t segments id the
p la te . T h e lo o p s h o u ld be fla m e d and c o o le d
between the different sets o f streaks. O n incubation,
g ro w th m ay be c o n flu en t a t the site o f o rig in a l
inoculation, b u t becomes progressively th in n er, am i
w e ll separated colonics are obtained over tbe final
series o f streaks.
T h e Jam? or carpet oj^fure provides a u n ifo rm
surface grow th o f the bacterium urn! is useful for
b s c ic n u p h a g e ty p in g and a n tib io tic s e n s itiv ity
te s tin g (disc m e th o d ), I t m ay also bo e m p lo y e d
w hen a large arruvunt []f g ro w th is required on solid
m edia as, for instance, in the preparation o f bacterial
.Lodgens am i vaccines. Law n cultures are prepared
by flooding the surface o f the plate w ith a liq u id
culture or suspension o f the bacterium , p ip e ttin g
o f f the excess inoculum and incubating the plate,
A lte rn a tiv e ly , the surface o f the plate m a y be
inoculated hy applying a swab staked in tbe bacterial
culture or suspension.
The stroke L-dJrtireii made in tubes containing
agar .slope (xhint) and is employed for providing a
pure growth of the bacterium tor slide agglutination
and other diagnostic tests.
Stab cultures are prep ared hy p u n c tu rin g a
suitable m e d iu m such as n u trien t gelatin or glucose
agar w ith a lo n g , s tra ig h t, charged w ire . T h e
m e d iu m is allow ed to set, w ith th e tube in the
upright position, p ro rid in g a flat surface at ih c IOp
o f the m edium - Stab cultures are em ployed m ainly
to r dem onstration o f gelatin liquefaction and oxygen
tequ i rcnmeiu o f tbe h a tre d urn under study. T h e y
are also used in the m aintenance o f stock culturcs.
F o r p r e p a r in g p e m r plate culture, tu b e s
containing 15 m l o f the agar m edium are m elted
and le ft to cool in a w a te r b a th a t 4 5 - 5 0
A p p ro p ria te dihitfons, o f the inoculum (o f I m l)
arc added to the m olten agar, m ixed w ell and the
contents o f the rubes poured into sterile Petri dishes
and allow ed to set. A fte r ineubatioin, colonies w ill
be seen w e ll distributed th ro ugho ut the depth o f
C o p y rig h te d m aterial
40 i Textbook d MicroBintogy *
iIn- medium and can be enumerated using colony
counter*. The pour plate method gives an estimate
ot’the viable bacterial count in a suspension and is
the recommended method for quantitative urine
cultures.
In the sweep plate method, the edges of the
Petri dishes containing the culture medium arc
rubbed over the fabric, with the medium Fa< ing it.
'Hie dust particles srurcd up from the cloth settle
on the culture medium, and colonics develop on
incubation. Thev can be counted and emulate*
made.
Liquid eu/nmes in tube*, bottles ot flasks may
he inoculated hv touching with a charged loop or
by adding the inoculum with pipettes or syringes.
Large lima:l.i can be employed in Liquid Cultures
and hence this method is adopted for blood culture
and for sterility tests, where the conccrtrarion of
bactcri a in the inocula is expected to be small. Liquid
cultures are preferable for Lnocula containing
antibiotics and other inhibitory'substances, as these
arc rendered ineffective by dilution in the medium.
Liquid cultures are also preferred when large yiclds
are desired, the yield being enhanced by agitation,
acral ion, addition of nutrients and removal of toxic
metabolites (continuous culture methods), 'llic
maim disadvantage of liquid culture lh tbat it does
not provide a pure culture from nixed inncula.
A n ib r o b il ; C m t i r e M e t h o d s
A naerobic bacteria differ in their requirement of
and sensitivity to oxygen. Some, such as Cl.
fijjtojyfjcrml, are aerotulerant and may produce
some growth on the surface of aerobic plates, while
others such as Cl- refunJ, are strict anaerobes and
form surface growth only if the oxygen ren-ion is
less than 2 mmHg. A number of methods have
been described for a ch ie vin g anitmbiosis, hv
exclusion of oxygen or production of a vacuum,
displacement of oxygen with other gases, absorp: ion
of oxygen by chem Leal or biological mean;;, and
reduction of oxygen.
1. Cultivation in vacuum was attempted by
incubating cultures in a vacuum desiccator, but
the method is unsatisfactory as some oxygen is
always left behind. Fluid adtures may hoi! over
and the media may get detached from the plates
in the vacuum produced- This method is not in
use now.
2. Displacement of oxygen with gases such as
hydrogen, nitrogen, helium or carbon dioxide
is somerimes employed, hilt thi-- method rarely
produces complete anacrobiosis. A popular, but
ineffective method is the caudle jar. Here
inoculated plates are placed inside a large
airtight container and a lighted candle kept in
i t hetore the 1i<1 is sealed. The bum ing candle is
expected to use up all the oxygen inside before
:l extinguished, but some oxygen is always
left behind. The candle jar provides a
concentration of carbon dioxide wh: cb
stimulates the growth of most bacteria.
3. In the chemical or bi<iloguaJ method, alkaline
pyrogalloi absorbs oxygen. This method, first
introduced by Buchner (1 8 8 8 ). has been
employed with different modifications for
providing anaerobinsis. Pymgallic add added
to a solution of sodium hydroxide in a large tn t
tube placed inside an airtight jar provides
anaerobiosis but a small amount of carbon
mono-ride, which is formed during the teat riun,
may be inhibitory to some bacteria. The method
has been applied 6o single tube and plate cultures.
The Spray anaerobic dish is a glass dish with
its bottom partitioned into two halves, tlie cop
accommodat ing half of a Petri dish carrying the
medium. Pyrngallic id and sodium hydroxide
are placed in the separate halves at the bottom
of the dish. The Inoculated culture plate 1*
inverted on the top of the dish and is sealed
completely. The dish i-s thru rocked to mis the
reagents, producing anaerobinsis. The anaerobic
dish is not in use now.
A si mplc modi fixation coos Lts of a Pcti i di^h,
between the two halves of which is inserted a
metal disc of tlightly larger diameter, with a
C o p y rig h te d m aterial
 « Culture Mrthpdi *
hole in the centre. T h e metal disc is attached to
the bottom half o f the Pfetri dish wirh plasticine.
Thro ugh the central hole , l few ]'diets o f fiddium
h yd ro xid e and 1 0 nil o f .i 1 0 % so lu tio n o f
pyrogallic acid are added. The inoculated halt
Of the Petri dish ! h then inverted <m the rnrtal
disc and scaled tightly.
The method in common use employe a disc
of filter paper having the same diameter as a
lVtri dish. If is placed on tup of ore half of the
dish and a mixture of pyrogallol and sodium
carhcmatc, in dry powder form, is spread on it.
The inoculated plate is inverted over the filter
paper and scaled tight with molten wax. The
dry pyrogallol mixture is activated by the
moisture within the eluded sys-teruj .and combine
anaernhiosls develops within about two hours.
Instead of alkaline pyrogaUol, anacnohtosis
lias Keen produced within jar*: with a mixture
nt chromium and sulphuric acid vRosenrh.il
method) or with vellow phosphorous.
Absorption of oxygen from small closed
systems has been attempted by incubation along
w ith aerobic bacteria, g trm in a lin g >eeds or
Fig. i t HcMm Ii-FIMw |ar
41
chopped vegetables, An&erobiosis produced by
such biological methods is slow and ineffective,
The most reliable and widely used anaerobic
method is the McIiiiath-PHded anaerobic jar
(Fig* 5.1). It consists of a stout glass or metal
jar with a metal lid which can be damped air
tight with a screw. The lid has two ruhes witli
taps,, one acting as the gas inlet And the other as
the outlet. The lid also has two terminals
which can be connected 1o an electrical supply.
Leading from the terminals and suspended by
Stout wines on the underside of the lid is a small
grooved porcelain spool around which is
wrapped a layer of pallid ini ted asbestos.
Inoculated culture plates are placed inside the
jar, with the medium in the- huttom half of the
plates, and the lid ckmpfid fight. The outlet tube
Is connected to a vacuum pump and the ait
inside is evacuated. The outlet tap is then closed
and the inlet tube connected to a hydrogen
supply. After the jar is filled, wirli hydrogen, the
electric terminals arc connected to a current
supply so that the palladlmsed asbestos is heated.
Th is acts as a cataivst lor the combination of
hydrogen with the residual oxygen present in
the jar. This method ensures complete
macrobiosis but carries the risk of explosion,
which may rarch occur. This risk can he
eliminated by modification of the catalyst.
Alumina pellets coated with palladium in u
gauze sachet suspended tnrm the lid of the jar
act as ai catalyst at room temperature, at long a*
the sachet is kept dry.
The Gtspak is now the method of choice
for preparing anaerobic jars. The Gaspak is
Commercially available as u disposable envelope,
containing chemicals which generate hydrogen
and carbon dioxide or the addition of water.
After the inoculated pi.Lies are kept in the iar,
rhe Gwpik envelope, with water added, is placed
inside and the lid screwed right. Hydrogen and
carbon dioxide are liberated and the presence
of a cold catalyst in the envelope permits the
C o p y rig h te d m aterial
42 i Textbook ef Microbiology *
combination of hydr<>Bjcn and oxygen to produce
an anaerobic environment. The Gaspuk is
simple and effective, eliminating the need for
drawing a vacuum and adding hydrogen.
An indicator should be employed for
verifying the anaerobic condition in the jars.
Reduced methylene blue is generally used fur
till1- purpose. It remains colourless anaerobically
but turns blue on exposure to oxygen.
4, Reduction of oxygen in the medium is achieved
by the use of various reducing agents, including
1% glucose, 0.1% thinglycolate, 0.1% ascorbic
a< id and 0,05% cysteine. Broth is an easily
prepared anaerobic medium into which pieces
of red hot metallic iron ate introduced. It is
then tavered over with sterile vaseline. Broth
containing fresh animal tissue, such as rabbit
kidney, spleen, testes or heart (Snurh-Noguchi
m edium )r supports the growth of many
anaerobes.
Thiogiycolate broth with hemin and vitamin, K
is an enriched liquid medium Tor culturing
anaerobic and microaerophilic bacteria.
Addition of a small quantity of agar enhances
the anaerobic capflv icy of the medium by slowing
the diffusion of oxygen in it.
Robertsons cooked meat medium is probably
the most widely used fluid medium for the culture
of anaerobes. It consists of fat-free minced cooked
meat in broth, with a layer of sterile vaseline over
it. If permits the growth of even strict anaerobes
and indicates their saccharolytic or proteolytic
activities, In the meat being turned fed or black,
respectively.
For fastidious anaerohes, particularly for
quantitative Cultures., pre-reduced media and an
smaertihiL; chamber ('glove bos’) may be used. The
anaerobic chamber is an airtight, glass-fronted
cabinc[ filled wilh inert gas, with an entry lock fur
the introduction and removal of materials, and
gloves for the hands.
M eth o d s oi Iso l a t in g P ure
C t LTUREs
The following methods may be employed for
isolaung pure cultures of bacteria from mixtures:
1. Surface plating is the method routinely
employed in clinical bacteriology and enables
the isolation nf distinct colonies- which may he
picked out, it'necessary' for further purification
and study.
2- Enrichment, selective and indicator medin are
widely used for the isolation of pathogens from
specimens such as feces, with varied flora.
fi. Pure cultures may be obtained by pre-treatment
of sp ecim ens with appro priate bactericidal
substances which destroy the unwanted bacteria.
This method is the standard practice for the
isolation of tubercle bacilli from sputum and
other clinical specimens, by treatment with
alkali, acid or other substances to which most
LummensaJ& are susccpLible but tubercle bacilli
□re nc-iRtant.
4. Obligate aerobes and anaerobes may be
separated by cuhivalion under aerobic or
anaerobic conditic ins. Shake cultures in Veillon
tubes were in use formerly but are now obsolete.
This consists Df a glass tube open at both ends-
O re end is closed with a rubber stopper and
molten glucose agar in which the inoculum is
evenly dispersed is poured into the tube and
allowed to set in a vertical position. The top of
the tube is closed with a cotton plug. On
incubation, the bacteria in the inoculum
differemiare depending on their oxygen
requirement- The obligate aerobes grow at the
cop and the anaerobes at the bottom, while the
facul tati'.re hacterin grow throughout the
column.The enrin! medium can be extruded on
to a pl.i.te and rhe different colonics fished out,
5. Separation of bacteria with different
temperature oprima can be effected by
incubation at different temperatures. Only
C o p y rig h te d m aterial
 ^ CuHura MfllhOdS ► 43

thcrm opli lie bacteria grow at fiO "C . A mixture
Centraining N. m eningitidis and N . catarrhalis
can be purified by in cu b a f'in at 2 2 lJC- when
only the latter grows.
6. F v heating a mixture containing vegetative and
spore form; ng bacteria, at SO the former can
be elim inated. T h is m ethod is userid for the
i sola tie r o f tetanus Luc i Hi fm m dust and sin i ilar
sources.
7. Separation o f m otile from nnnmotile hacDeria
ta n be effected using CtAigii is lute. T h is consists
o f a tube o f sem i-olid agarh with a narrow tube
open at hoth ends placed in the centre o f tile
medium in such a way that it projects above the
Level o f the m edium. T h e mixture is inoculated
into the central tube. O n intubation die motile
bacti-ri.L alone traverse the agar and appear at the
top o f the medium out-ide the central tube.
AU-tuhc also serves the same purpose, inoculation
being performed in one limb and the subculture
taken from the other. This method can also be
used to obfci i1 phase variants in Salmonella species.
8. F a th o g e n ic b a cte ria m a y be isolated from
mixtures by iooculaLiun intoappropriate animals.
A n thrax bacilli can be distinguished from other
aerobic sporulatiing bacilli by inoculation into
mice or guinea pigs. A nthrax bacilli produce a
fatal septicemia and may be cultured pure from
the bean blood.
'■>. Bacteria of differing sires may be separated by
the use of selective fdters. Filters are widely used
for separating viruses from bacteria.

F u rth e r Kvudinj*
ColleeJG er ii. 19%. ftaa.-cW Medical Microbiology. 4'1 edn. Edinburgh; Ch.urch]]l L iv iigitone.
Murray PR et al. 1999. Manuai o f Clinical Microbiology. 7“ edn. Wa&lu'gronc DC: American Society of Microbi<'loRy.

C o p y rig h te d m aterial
 Identification of Bacteria
O n ce a bacterium has l^ccn obtained m pun; culture,
ith s s t n be id en tified .Th e following characteristic:;
have to he studied in the process.
The morphology ut the bacterium depends on a
□umber of factors such its the strain studied hnature
of i In' culture medium, temperature and time of
incubation, age cit Hll: culture and the number of
subcultures it hits undergone, The characteristics
noted arc shape, skm, arrangement, motility, flagella,
spores and capsules. All these cannot he made out
in a single medium. The shape may he spherical„
filamentous, rod shaped, comma shaped or spiral.
The axis flf the organism mav be straight or curved.
The length and breadth may vary. The sides of thr
organism may be parallel, convex, concave or
irregular. Tbe ends- iii.lv be cut straight, rounded or
tapering. Considerable variations in rbape and size
leading to club, navicular and swollen or shadow
or giant forms may be seen. They may be arranged
singly, in pairs, in tetrads or in packets of eight, or
in chains, short nr lung, in thr cast of cocci; bacilli
may he arranged at ra ndom, in siion or long chains,
silChinese letter patterns, as palisades or in bundles;
vibrios may he single nr in S or spiral forms, They
may be normiotile, dtiggishly motile, actively motile
or may exhibit darting motility. They may be
without flagella, that is atrirhatc, or nHmotrichatc,
lo]ihotriehate, ampbit debate or pcritrichxte. The
spares, when present may he oval or spherical or
ellipsoidal and may he o f rhe same width or wider
than that oI the bacillary body, The spores may be
equatorial, suhtemninal or terminal, (\ipsulcs may
or mav not he present. Hanging drop preparations,
- Lark gnou nd iILumioatiftn, phase eon trust or electron
microscopy all help in these studies.
The age of the culture l& important. In older
cultures, staining characteristics either vary or are
n.ol hrought out well. Simple stairs bring out the
morphology best. Differential and special stains ait
necessary to bring uui chancteriutict like flagella,
capsules, spores lull! metachromatic granules. The
<rram sruin divides bacteria into Gram positive and
Gram negative; the Zichl-Neclscn stain into acid
fast and non-add fast. The fluorescent antibody
technique enables one to identity them according
tn their surface antigens.
The study of morphology and staining
characteristics helps in preliminury identification
ut'the isolate.
C]|^. p,/-. jvgjgff'i
These provide additional information for the
Identification of the bacterium. The characteristics
revelled in differe nt tj^ ^ t.jf media are noted. Wh ile
studying colonies on solid media, the following
features are noted;
1. shape— circular, irregular, or rhizoid;
2 . size in [nllLmetres;
3- elevation—effuse, elevated, convex, concave,
umHunate or umbilicatc;
4. margins— bevelled or otherwise;
i. surface— smooth, wavy, rough, granular,
papillate or glistening;
C o p y rig h te d m aterial
6- edges—truing undulate, erenated. fimbriate or
curled;
7. colour;
S. structure— opaque, translucent or transparent,
9. consistency— mem bra ntn.it, friable, bulyniVt ur
viscid;
III.emuJi-iliability; and
11.whether they are differentiated into a central
and a peripheral portion.
In a stroke culture, note
1. tire degree of growth—scanty, moderate, or
profuse;
2- their nature— discrete or confluent, filiform,
spreading or rhizoid;
."3. their elevation, surface, edges, colour, structure,
odour, CITVULsifiability, consistency and changes
in the medium.
In :l fluid medium, the degree of growth,
presence of turbidity and its nature, presence of
dcpjhit and its character, nature of surface growth
Mich as pci Licit? and irs quality and ease of
disintegration, and odour -are noted.
The resistance of the organism to heat .Liui to
disinfectants is rested, both for vegetative and spore
forms. The resistance of 5. &tcalis heat at 60 "C
for half an hour and of clostridial spores to Soiling
for various periods are examples. Resistance to
antibiotic and chemotherapeutic agents and
h.LcDenocios would also help in differentiation and
identification.
The requirements of oxygen, the need for carbon
dioxide, the capacity to form pigments, and the
production of hemolysis help in classification.
The more important and widely used rests are
described below:
This is tested in sugar
media. Acid production is shown hy change in the
colour of the medium to pinh or ted, and the gas
produced collects in Durham’s tube.
There may be no change in
the medium, or acid or alkali may be produced;
clt HtTing of mi Ik, peptoi lisatiLut or s.iponificn Lkul may
occur. The clot miay be disrupted by rht gas
produced (stormy fermentation).
T h is is tested In a
p ep to n e w ate r c u ltu re after 48 o r 9 6 h o u rs
incubation at .17 "C . This test demonstrates the
production of indole from tryptophane. Add
0.5 ml Kovac’s reagent and shake gently. Red colour
indicates a positive reaction. Kovac's reagent
consists of
PalbdinlethvLaniinobenz aldehyde b) g
Amyl w isoamyl alcohol 150 ml
Concentrated HC1 50 ml
This is prepared in smafl quantities and stared
in the refrigeratin'.
This Lest is
employed! to detect the production ot acid during
the fermentation of glucose and the maintenance
of a pH below 4,5 in aii old culture. Five drops of
0.04% solution of methyl red are added to rhe
culture in glucose phosphate medium which had
been incubated ar 30 “C for five days, mixed well
and read at once. Red colour is positive while yellow
signifies a negative test.
This lest
depends on the production of acetyl
methykaibinol from pyruvic acid, as an
intermediate stage in irs conversion to 2:3 butylene
glycol. In the presence o f alkali and atmospheric
oxygen, the small amount nt acetyl methyl caibinol
present in the medium is oxidised to diacetyl which
reacts with the peptone of the bmth to give a red
colour.
r|Tie test is jwrformed by adding 0.6 ml o f a 5 %
solution o f ft naphrbol in ethanol and U.2 ml o f
4 0 % K O l l to one ml of a glucose p ho sp hate
m edium culture o f the organism incubated at 30 "!C
fur five lI uvs or M "C I nr 48 houts. hi a posilrvic
C o p y rig h te d m aterial
 reaction., a pink colour appear* in 2 -5 minutes,
deepening to magenta nr crimson in half an hour.
J]i a negative reunion, i[ remains colourless for half
an hour Trices of pink colouration should he
ignored,
Koscr's citrate medium
has citrate as the sole source of carbon. Ability to
yse this substance is indicated by the production nt
turbidity in the medium.
Indole, MR, VP and citrate teals are very useful
it) the identification and classification of enteric
Cram negative bacteria. These tests arc commonly
referred to by the sigfa TMVjC 1 tests.
This is tested after
growing the bacterium for five days at 37 aC in a
broth containing 1% KNOr The test reagent
consists lit'a mixture of equal volumes of solutions
of sulphanilir acid and ct-naphthylaminc in 57 Not
acetic acid mixed just before use. 0.1 oil of the test
reagent is added to the culture. A red colour
developing within a few minutes ^igioifies a
positive reaction, while absence of colour indicates
a negative reaction. This is a test for the presence
of the CtBynte nitrate reductase which reduces
nitrate to nitrite.
Tu a peptone
water culture grown for live days at 37 4C >Messier's
reagent is added. Hrown colour is positive and faint
yellow colour negative.
This test is done In
Christensens urease medium. Inoculate the slope
heavily and incubate at 37 "C, Examine after four
hours and after overflight mentation. The tevt
should not be considered negative till after four
days of .incubation. Urease positive cultures produce
a purple pink colour. Urease producing bacteria
reduce urea to ammonia which is responsible for
the colour.
Some
organ i-sms decompose sulphur-containing
aminoacids producing H S among the products.
When cultured in media containing lead acetate,
they turn them black or brown, Instead of lead
acetate, ferric ammonium titrate or ferrous acetate
can be used. The organisms can he grown in
culture tubed Between the cotton plug and the
tube insert a filter paper strip soaked in 10% lead
acetate solution and dried. Browning of the paper
indicates H ,$ production.
One drop
of 1% aqueous methylene blue in added to the
broth culture, and incubated at 37 “C. Complete
deculourisaiion is strongly positive, while green
colour is wealth' positive.
Place a Loopful
H jO , on colonies on nutrient agar. Prompt
effervescence indicates catalase production.
Culture media containing blood are unsuitable for
the test as blood contains catalase.
I bis reaction is due
to a cytochrome oxidase which catalyses oxidation
of reduced cytochrome by oxygen. A 1 .0 - 1.5%
solution nf tetra methyl p-phcnylcnc diamine
hydrochloride is poured over the colonies. Oxidase
positive colonics become maroon, purple and black
in 30-30 minutes. H ie test can also lie done by
Kovac's method, A strip of felter paper, soaked in
rhe oxidase reagent i,s placed in a fttri dish and the
L-utuny to be tested is smeared on the paper in a
line about 5 mm long. In a reaction the smeared
area turns dark in 10 seconds. The solution should
be freshly prepared.
Organisms producing
lecithinase {for example, Cl- perfringens) when
grown on a solid egg yolk medium, form colonics
surrounded by a zone nf clearing.
Buffered
Liquid medium containing KCISr in a final
concentration of about 1/13,000 is used to identity
some RUN tolerant enteric bacilli.
are being used
increasingly for the identification nf isolates. These
are convenient and economical, as a single composite
medium indicates different properties of the
bacterium which otherwise would have required
the use of many separate media. A popular
C o p y rig h te d m aterial
composite medium is the Triple Sugn Iron (TS1)
medium which indicates whether a bacterium
ferments glucose only, dr lactose and sucrose also,
with or without gas formation, besides indicating
I I .S production as w ell. The medium is
distributed, in Cubes, with, a hurt and slant. After
Inoculation., i1 the slant re mains red and the butt
becomes yellow, all [Lie Sugars - glucose, lactose
and sucrose - are fermented. Bubbles in the butt
indicate gas production and blackening of the
medium shows formation of H 2S. The T S l
m edium facilitates prel im in arc identiIncation of
tira m negative bacilli.
Other tests such as fermentation oforganic acids,
oxidation of gluconate, aminoacid decajhodfykrijOrt,
and hydrolysis of sodium hippuracc arc sometimes
employed. With increasing knowledge of [lie
metabolic processes in the growth of various
bacteria hrhe number of tests too is on the increase.
Special manuals have to be consulted for die details
and utility of these tests.
By using specific sera
wc can identify organisms by agglutination or other
suitable serological reactions. Immunofluorescence
test is useful in some cases.
These enable intnispecies typing of some bacteria,
Pathogenic] ty teats by
inoculation of the test organism into laboratory
animals like the guinea pig, rabbit, rat and mouse
by intruders id, subcutaneous, intramuscular,
Lntxaperitooeal, intracerebral or intravenous, or bv
oral or nasal Spray were common procedures for
identification ofisolatcs in the past. They are rarely
used now because simpler in vitro tests arc available,
While classical phenotypic characterisation of isolates
takes days, automated methods are now available
which only take hours. I dm I if]cation is simplified
by the detection of specific enzymes, owdns, antigens
or metabolic end products o f the isolates. For
example, mimy obligate anaerobes can be identified
nip idly by gas liquid chromatography o f tile short
chain fattv acids pnaluced bv them during glucose
fermentation. M olecular methtjds such as polymerase
chain reaction and other amplification procedure;
coupled with nucleic acid probes carrying specific
D N A or RNA bast sequences arc now widely used
for identifying microbes.

t ’nvrju S T ed- 1978. fX'oMflJUy (d'M&iiwI TVtfiFKuny I -cnJwn Oxlbn.L University Ptex*.
Cowan ST ind KJ Steel 1 % 5 . AJanuaJ/iir the Idcniificatkm o f M edial Bacnerfa. London. Cambridge University Press.
Fmegold SM and EJ baron 1986. Baikyand Scoff's DhgnoiZic Bicteriolog)'. 7* edn St,Louis: C V Mosby.
GnodfeLlnw M and R G Board ed. 198U. AficroiNi Jc^jk-aJ Classification and Identification. London: Acadennc Prera.
Murray P ef ul. ed*. 1999. Ma/tuaf o f CJi'jika/ AfH.mbeokgy. 7lk edn. Washington: American Soeietv for Microbiology.

C o p y rig h te d m aterial
Bacterial taxonom y or systematics m m prists three;
components:
1, Classification, nr the orderly arrangemctir of
units. A p o u p o ftrnit?isdlcilx t™jij(pl r.j.vji),
irrespective of its hierarchic Level.
2. Idm [i furi[ion of an unknown with ade fined and
named UMt* acid
Nomenclature, nr the naming . it units.
For purposes, ofcbuificidon, ii i* necessary ro
determine .lh many characteristics of bacteria as
po^siLilc. Such characteristics may be weighted,
greater imporUince luring given to «)mc than to
others, or they mav he assigned CL|ual importance,
depending ori the method of class!Ilealmn. On the
contrary, hir purposes el identihing bacterial
isolates, it is important ro devise a kty using the
minimum number of important characteristics
which can he easily tested.
The first attempt!
at bacterial classification (Mueller l7$b;
iihrcnbcrg 1®3£) were made when Little was known
j Limit bacteria. Haeckel (l®6-f>) classified all
unicellular organisms as Protista. Cohn (1872-75)
nude .i inorjifioLogic.il classification, integrating
tiaetcria with the blue-green algae in the class
Sdiizophyta. A detailed system of classification was
projHJsedi by Migulu ( 1®V4). As more inturnuban
became available on the physiologiesI and
biochemical properties of bacteria, these were
employed in proposing new systems of bacterial
classification by Knight (193b), KJuyver and van
Niel (1936) and others.
Bacterial classification presents special problems.
Linnaeus (1735) divided! all living LM-tngs into two
kingdom-., pi nut and animal. Bacteria had been
placed in the plant kingdom and designated as
6Vhjzomycert? (fission fungi). But ns bacteria
present h“abires common to both plants ami animals,
it has been proposed that a new kingdom. Moncia,
he created, to accommodate all microorganisms
without true nuclei, plaltids and sexual
reproduction (Stanicr .md van Niel 1941), This
proposal has not met with universal acceptance.
K ingdoios are divide*t sucecssivcly into *tivi sion,
class, order, family, uibt. genus and species. For
example, the full taxOtiqmica] position of the
typhoid h.Lcillu?. is as follows:
Ohfsjor? Ptoiophrta
O ais Sch ip.oi nyce ten
Order Eubacrenales
Family En tEnptactBRKWH
Tribe SaljUone llae
f Jenna Salmonella
Spheres Salmonella ryphi
Species
is the standard taxonomicil unit in biology'. With
higher forms of life, a sjiecies unit constitutes a
stage ofevulutiun, with a charatteristii.: morphology,
and is delimited by the failure of interbreeding
outside the unit. But in bacteria, 1 lit species concept
is vague and ill defined. 1hie to the absence of foss-il
remains in bacteria, the evolutionary status of
spc'.'its cannot be established Morphological
differences are insufficient fen the definition of
bacterial Species. The general absence of sexual
reproduction in bacteria prevents the use of
inbreeding as a test for differentiation between
species.
C o p y rig h te d m aterial
 In Spite o f these difficulties* the concept o f
species provides a co n v en ien t unit in bacreriid
taxonomy. Resides m orphological features, criteria
useful for the d e fir itio r o f bacterial species arc
p h y s io lo g ic a l, b io c h e m ic a l, a n t ig e n ic and
path o g en ic properties. A s 'sp ed es' is a genetic
concept, definitive information can be obtained by
com parison o f the nucleotide base ratios, w hich
are constant for any one species but may he different
in different species. G e n e t ic hom ology can be
dem o n strated by D N A h y b rid iz a tio n between
d iffe re n t in d iv id u a ls o f the sam e sp e cie s.
C o m pariso n o f r R N A sequences helps to arrange
bacterial species into a phylogenetic tree. 1 6 S r R N A
s e q u e n c in g has e m erg e d as u se fu l to o l for
identifying m any new unculturahle pathogens (e g .
Bartonella h cn sch c).
An im p o rta n t d iffe re n c e b etw e en the
c la s s if ic a tio n o f b a cte ria a n d th at o f h ig h e r
organism s is that in the former, the properties of a
population are studied, and not o f an individual. A
population derived by binary fission t o m a single
cell is called a clo n e . A single bacterial co lo n y
represents a clone. T h o u g h all the ceils in a clorw
arc expected to be identical in ail respects* a few o f
them may show differences due to m utation. A
population o f bacteria derived from a particular
■ ounce, such as a patient, is called a strain.
T h e genera] absence o f sexual reproduction in
bacteria serves to maintain their character OOJlStant-
B ut bacteria possess several features chat contribute
to sonic degree of herciugvneity in dieir jmpulaiLons.
T h e ir short generation time and high rate o f mutation
lead to tlur picscncc, in any population* ut cells with
altered character. Methods ofgeitetii: exchange such
as transformation* transduction and conjugation cause
differences in character. Prophage and plasmid D N A
COO induce new properties.
T h e re are two
a p p ro a c h e s to b a c te ria l c la s s if ic a t io n . T h e
hierarchical classification represents a branching
tree like arrangem ent, one characteristic b e in g
employed for division at each branch or level. T ills
system is called phylogenetic because it implies urn
evolutionary arrangement o f species. H e re some
c h a r a c te r is t ic s are arbi rra rily g iven s p e c ia l
weigh rage, D e p e n d in g on the characteristic so
chosen, the cla ssifica tio n w ould give different
patterns. For example, the intestinal G ra m negative
bacilli have been traditionally classified d e lu d in g
on whether they ferment lactose or not. W h ile this
provides a useful distinction between the pathogenic
and non p ath o g e n ic groups o f these b a c illi, a
di fie rent hut useful classification could be obtained
using form er ration o f sucrose as the crite rio n .
W h ile c la s s if ic a tio n b ase d or: w e ig h te d
characteristic is a convenient m ethod, it has the
serious drawback that the characters used m ay nor
he valid. Ferm entation o f lactose* in the example
cited* is not an e sse n tia ! an d p e rm a n e n t
characteristic. It m ay he acquired or Inst, upsetting
the system of arrangement.
T h e A d a n so n ia n
classification, so called after M ichael A dausnii who
introduced it in the eightenth century, avoids the
use o f w eighted c h a ra c te ris tic s . It m akes no
phylogenetic assum ption bur m erely takes into
account all the characteristics expressed at the tim e
o f study. H e n ce it is called a phcncric system. It
gives equal weight to all measurable features-, and
groups organism s on the basis o f sim ilarities o f
several character istics. T h e availability o f computers
has extended the scope o f phenetic classification
by permitting comparisons o f very large numbers
o f properties of several organisms at the same rime.
11m . is known as mrmen'ca/ taxonomy.
T h is
is based on the tfegree n f genetic rdatedness o f
d iffe re n t o rg a n ism s, b ir c c a ll p ro p e rtie s arc
u lt im a t e ly based on the genes present* th is
cla ssifica tio n is said to be rhe m ost natural or
fundamental method. D N A relatedness ran he
tested by studying the nuclei.it ide seque uces o f 1 1N A
and by D N A hybridisation or recombination, methods.
T h e nucleotide base com position and base ratio
(Aden in e -T h y n iin e iG u a n in e —Cytosine ratio ) varies
C o p y rig h te d m aterial
widely among different groups ot microorganisms,
though it is constant for members of rhe name
species. Molecular classification has; been employed
more with viruses than with bacteria,
No method of bacterial classification in
universally accepted. The method most widely
adopted is presented in successive editions of
B ogeys jV/arrua/ 0/ Oetenrpinatrve Bacteriology'.
For diagnostic
or epidemiologic^] putpuso, it is often necessary ro
subeksrify bacterial species, This may be hased on
tijcKhemiL-.Ll pniperties [biertypet), antigenic fi'amres
(serotypes), bacirefioph age sasccptibiliiy (plu^e [>>*:>)
or production ofbacwri<icins (colidn types), A species
ma()r he divided first into groups ^nd ibun into Cv|ies,
as for example, in streptococci.
Much greater discrimination in iittraspecics
typing has been achieved by the application of
newer techniques from immunology, biochemistry
and genetics- Jnvtstigarions ol epidemiolngy and
pathogenesis using these techniques have been
collectiveIv referred to as rMoJecuiar tfpjdeimoJoify,
The methods used sre ot two types: phenotypic
(study of expressed characteristics) and genotypic
(direct analysis of genes, chromosomal and
ettnu-hrnniusonial DNA). Molecular phenotypic
methods include electrophoretic typing of bacterial
proteins and immunoblotting. Genotypic methods
include p la s m id profile ai]jlysi&, rest fiction
endonuclease analysis of chromosomal DNA with
Southern blotting, PCK and nucleotide sequence
analysis. Som e ot these techniques are considered
in the chapter Bacterial Genetics,
The need tor applying generally accepted niitieS
for bacterial species is self-evident. Chaos will result
if the same bacterium is referred toby different names
by different workers. Internationa! agreement on
bacterial nomenclature 3* ensured by rhe Code of
Nomenclature which has the auihoiity of I lie
International Association of Microbiological Societies.
Two kinds ol' names arc given to bacteria. The
first is the casual 01 common name which varies from
country to country'and is in the local language. Names
such as typhoid bacillus' and 'gonococcus' arc casual
names. Such mimes are useful for communication
the local level. The second is the scientific or
international name which is the same throughout the
world. The scientific name consists usualLv ot two
words, the first being the name of the genus and the
second the specific epithet (for example Bacillus
subriiis}. The generic name is usually a Latin noun.
The specific epithet is an adjective or noun and
indicates some property of the species (for example
iJihu.s meaning white), the animal in which it is
found (for example sujs, means pig), the disease it
causes (tetaur, of tetanus), the person who
discovered it ( ueJchtj, after Welch) or the place of
its isolation { le> ndon)< The genetic name always
begins, wirh a capital letter and the specific epithet
with ,1 small letter, even il it refers to a person or
place (for example Salmonella louden).
As, a point of reference, type ailtures of bacteria are
maintained in intcrnatiHinal reference UxgaJforicSrThc
type cultures contain representafiv« of all established
species, The origintil cultures of any new species
described are deposited in type collections- Ihey are
made available by rhe reference laboratories to other
workers for study and comparison.

C ow an S T ed. 1978. Dictionary ofMedics! laiun oray. London; O xford University I V es,
Cewan ST ;ind KJ Steel l9fo. Minus! tor zhe ldentiticsii\>n ciM ed n il ftaderii London; Cambridge University Press.
FiuvpiJd. SM and FJ Batun 14^^. Bliley and Scurfs Dj^jnusur HarterjoJogk. 71* edn. St. Louis; CV. Mmby.
Coodfcllrvw M and RG Board cd. 19SO. M icrobiohgiol ClmifidertHl and Identitic-ition. London; Academic PiesE.
Muttay f m j E, ads. ]9W . i'llsnua} itf'Ciiiircii Ali&obiology. “If edn. Washingnon: .American Society far MjciobuJogy.

C o p y rig h te d m aterial
 Bacterial Genetics
Generics, i name euined by the British bLo!ci^ist
William Bateson in 1906, is the study of heredity
dwd variation, keeking to understand the causes of
the resemblances and differences between parents
and their progeny. Like other organisms, bacteria
also breed true and maintain rheir characteristics
from generation to generation, yet at the same time,
exhibit variations in particular properties in a small
proportion of their progeny. Though her Liability
and variatinn in bacteria had been noticed from
the early days of bacteriology, it was not realised
then that bacteria too obey the laws of gciwties.
“V't frf;
Even the existence of a bacterial nucleus was a
subject of controversy. The differeneet in
morphology and other properties were attributed
by Nagcli (1677> to bacterial /tfeomotphram, which
postulated the existence of a single, or perhaps a
tew species of bacteria, which pitssessed a protean
capacity for variation. With the development and
application of precise methods of pure culture* it
became apparent that different types of bacteria
retained constant form and function through
successive generations. This led to the concept of
monomotjjhj'sm, proposed by Cohn and Koch,
which admitted of little potential for variation and
separated bacteria into species based upon single
character differences.
U was only since the 1940s that principles of
genetics were applied to bacteria and their viruses.
I"his has led nor merely to a better understanding
of die genetic processes but also to fundamental
advances in biology rid biochemistry and to the
11: rrh ot\i new hranch of science, molecular biology.
B asic P r in c ip l e s o f M o l e c u l a r
B io lo g y
The ‘central dogma" of molecular biology is that
deoxyribonucleic acid (D N A ) carries genetic
information, which is transcribed onto ribonucleic
add i RK \ 1 .in.: then translated as the particular
polypeptide (D\A —^ R!SFA —Vpolypeptide). As
the nature and functions of a cell arc basically
determined by the pecific polypeptides that
constitute il- proteins and enzymes, it is evident
that the essential materia] of heredity is DNA
which is the storehouse of all information for
C o p y rig h te d m aterial
protein synthesis, (An exception exists in the case
r>t some viruses in which the genetic material is
RNA instead of D N A )
The DNA molecule is composed of Cwo chains
of nucleotides ■wound together in the form of a
'double helix’ (Pig. 8.1). Each chair has a backbone
of deoxyribose and phosphate residues arranged
alternately. Attached to each deoxyribose is one of"
four nitrogenous bates, [lie purines, adenine (A)
and guanine (G), and the pyrimidines, thymine (T)
and cytosine (C). The double-stranded namre of
[he molecule is stabilised by hydrogen bonding
between rhe bases nr the opposite strands in such
a manner that adenine is always linked to thymine,
anti guanine to cytosine (Fig. R2).
Adenine and thymine thus form a
cf hriiplemenrary base pj ir, and guj nitLe and cylusii ll1
form another. A molecule of DNA will, therefore,
contain a> many units of adenine as thymine, and
of guanine as cytosine but [he ratio of each pair of
bases [A 4 T) /(G + C), though constant for each
species, varies widely from one bacterial species; to
another The DNA molecule replicates by first
unwinding at one end to form a fork, each strand
of the fork acting as a template for the syr thesis of
a complementary strand, with which it then forms
a double helix.
Basically RNA is structurally similar to DNA,
except for two major differences. It contains the
sugar I'ibose (instead of deoxyribosi which is
present in DNA) and the base uracil (instead of
thymine which is present in DNA). Three distinct
types of RNA can be distinguished on the basis of
structure and (unction: messenger RNA (mRNA),
nbosomal R N A (rR N A ) and transfer RN A
(tRNA). DNA acts as the template far the synthesis
ofmRNA and, therefore, the bases in the two will
he complementary to each other. Adenine, guanine,
cytosine and uracil in mRNA will be
complementary to thymine. Cytosine, guanine and
adenine, respectively, in the DNA-
Genetic information is stored in the DNA as a
code, the unit of the code consisting of a sequence
of three ha&cs, that is, the code is triplet. Each triplet
(codon) transcribed on mRNA sjuniftes for a single
amitmarid hut the code is Regenerate' so that more

BACKBON h---- ^ |-------SIDE CHAIN-------j dc.

/
DEOXYRIBOSE ADENINE- -T H Y M I N E — D E O X Y R IB O S E
/ \
PHOSPHATE PHOSPHATE
\ /
D EO X Y R IBO SE G U A N IN E * - C Y T O S JN E — D E O X Y R IH O S E
/ \
PHOSPHATE PHOSPHATE
\ /
DEOX YRl BOSE CYTOSINE- -G U A N I N E D EO X YR I BO SE
/ ______________ \
etc.
ONE STRAN D

DOUBLE-STRANDED DNA-

C o p y righted m aterial
than (me codon may exist tor the same am inuacid.
Thus, the triplet A G A codes for arginine but the
triplets AGO, CGU, CGC, CGA and CGG also
code for the same a mi noacid. The code is non-
overlapping, each triplet being a distinct entity, and
no base in (me codon is employed as part of the
message of an adjacent codon. Three codons (UAA,
UAC and UGA) do not code for any aminoacid
and arc called 'nonsense codons*. They act as
punctuation marks (stop codons) terminating the
message for the synthesis of a polypeptide.
A segment of DNA canning codons specifying
for a particular polypeptide is called a gene. A UNA
molecule consists of a large number of genes. each
of which contain* hundreds of thousands of
nucleotides. T h e bacterial chromosome consists of
a double-stranded molecule of DNA arranged in a
circular form. When straightened, it is about 1,000
|Ltn in length. The length of DNA is usually
expressed as kilubases (1 kb =■ 1.000 base pains).
Bacterial DNA is about 4.000 kb and the human
genome about 3 million kb long.
h] higher forms of Life, several stretches of DNA
that do not appear to function as- codons occur
between the coding sequences of gents. These
Apparentlv useless noncodiug intm sinus are called
jiifrojis, while the stretches of coded genes are
called exuni;. During transcription* the genome is
copied in i.ts entirety; both introns and exons. The
introns ary then excised from the KNA copy before
being translated by the ribosome into proteins.
In addition to chromosomal DNA, most bacteria
possess dtCrachromosumal genetic elements. These
arc not essential for the normal life and functioning
of the host bacterium but may confer on it
properties such as drug resistance and trudgen icity
leading to survival advantage under appropriate
Conditions. P.l.i>njida are firclilat D N A molecules
present in the cytoplasm of bacteria, capable of
autonomous replication (jmfepejFiJejif rejilicrtirts). By
their ability to transfer genes ffOm one cell to
another, plasmids basic become important vectors
in genetic engineering. Plasmids may also be seen
in yeasts* which are eukaryotes. Plasmid DNA
same tunes may be integrated with L'liromcisnrnu]
DNA. The name epiznme was employed for such
integrated Tones, though this distinction is not
usually nude now.
Plasmids have been classified in many ways,
depending on whether they are self-transmissible
or nun transmissible (nanconjugating), on the
property encoded (sex, drug resistance, etc.), by
restriction endonuclease fingerprinting or other
criteria. An important method of plasmid
classification in by incompatibility typing. Closely
related plasmids do nor coexist stably in the same
bacterial cell, while unrelated plasmid-, can. On this
ba-us, plasmids have been classified into different
in co m p a tib ility g ro u p s . T h e y have also been
classi tied based on the types of conjugation tube
induced* which determine the susceptibility of the
host bacterium to lysis by some virulent
bacteriophages.
The sum total of the genes that make up the genetic
apparatus (it a cell (genome) establishes its genotype,
which is the heredity ry constitution of the cell that
is transmitted to its progeny. Thegcncnt'])c includes
the complete genetic potential of the cell* all of
which may or may not be expressed in a given
environmental situation.
The phenotype [phacnv meaning display) is the
physical expression nt the genotype in a given
environment. It follows* therefore; that a cell miv
exhibit different phenotypic appearances in different
situations; fur example, the typhoid bacillus is
normally flagellated but when gmwn in phenol agar,
the flagella are not synthesised. This is only a
phenotypic variation determined by the environment­
al Lii is reversed when snbcultured from phenol agar
into biflth. Another example of environmental
C o p y rig h te d m aterial
54 4 TexItjOQk Ol MiCrOiJiDlOQy 11
influence is the synthesis by E. l’oJj or the enzyme
betargalactosidasc, necessary for lactose
fermentation.’Ilie bacillus possessc* the genetic
information for the synthesis of the enzyme Hut
the actual synthesis Lakes place only when Lt is
grown in a medium containing lactose. When
grown in a medium containing glucose only, the
enzyme is not synthesised. Such enzymes which
are synthesised only when induced by the substrate
are called in d u ced enzym es* as opposed to
cefljfinjtjvf ens.rm cs, which are synthesised
irrespective of the presence nr absence of the
substrate.
Phenotypic variationh are influenced bv the
environment, Limited in range by the genotype,
temporary and not heritable. Variations are s;:.id to
be genotypic when they arc due to alterations in
the genome. Genotypic variations arc stable,
heritable and rot influenced by the environment.
They may occur by mutation or by one of the
mechanisms of geiicLk transfer or exchange, such
as transformation, rransducti-.m, lysogenic
conversion and cuciiugalion,
M u t a t io n
Mutation. is a random, undirected, lien table variaunn
caused by an alteration in the nucleotide sequence
at some point of the DNA of the cell. It may be
due to addition, deletion nr substitution of one nr
more bases (point mutaf.on)- Multiple mutation*
cause extensive chromosomal rearrangements. A
mrsscniT muiaiioit :a one in which the triplet code
is altered so as Co specify an .noim u, id ditrcrcut
from that normally located at a particular position,
iii the protein. Deletion of a nucleotide within a
gene may cause premature polypeptide chain
termination by genera[ing a nonsense oudnn, i.e.r
nonsense i n u t i i ion. 11 ii IstierHiurt ; ■■ substiiution of
a purine for a pyramidine and vice veraa Lil base­
pairing- Suppressor mutation is reversal of a mutant
phenotype by another mutation at a posi tion on
the DNA distinct from that of the original mutation.
Ah genes are susceptible to mutalioilal events buL
netball mutations sue expressed. Some mutations
Involve vital functions, and such mutants are
ntm viable {lethal mu ran on). A type of lethal
mutation which ls of great interest is 'conditional
mutation’- A conditional lethal mutant may be able
to live under certain conditions (permissive
conditions). The commonest type of conditional
mutant is the temperature sensitive (fit) mutant,
which is able to live at the permissive temperature
(say, .15 ^C), but not at the restrictive temperature
(say, 39 L'C).
Each gene undergoes mutation with a fixed
frequency. M utalio n rates ol individual genes in
bacteria range from 10^ to 10"K per bacterium per
division. The molecular mechanism of mutation is
that during DNA replication, some 'error7 creeps
in while the progeny strands are copied. For
initanec, instead of thymi :ic bond mg wh h aden i:ie,
it may, due to tautomerism, sometimes bond with
guanine. Though mutation occurs spontaneously,
: ts frequency can be increased by several agents
(muragemi) such as UV rays, alkylating agents,
acridine dyes, l-brnmnuracit and 2-amLnopiiriiire.
Mutation Is a natural event, taking place all the
rime at its particular frequent) in all the dividing
forms of life. Most mutants, however, go
un reengn ised as the mutation maybe lethal or may
affect some minor funcrlmn that may not be
expressed. Mutation is best appreciated when it
involves a function which can be readily observed.
For example, an } coJj mutant that loses its ability
to ferment Lactose can be readily detected on
MacConkey agar but is unrecognisable on nutrient
agar. Mutation is of vital importance when it
confers a survival advantage. If a streptomycin
resistant mutant of the tubercle bacillus develops
in a patient under Treatment with the drug, u
multiplies selectively and ultimately replaces the
original drug sensitive population of bacteria, Hut
in a patient who is not cm treatment, the mutation
confers no survival advantage and, therefore,
preferential multiplication of the mutant docs not
occur. Such changes in die character of bacterial
C o p y rig h te d m aterial
populations, observed in the presence of a selective
environm ent, were formerly considered! to be
'adaptations'. By a post hoc, ergo propter hoc
reasoning, the environment and the variation were
believed to have a eause-ind-cffect relationship.
Such induced variations were considered heritable
in the lamarckian sense, lr was the demolition of
this concept of adaptation in the 1940s that
established bacterial genetics on a firm scientific
basis.
The proof that bacteria undergo spontaneous
mutation independent ol the environment was first
provided by l.uria and D e lh n ih k (19 4 3 1 bv the
'ilnL’tTjation rest'. They found that very wide
fluctuations occurred in the numbers of
bacteriophage resistant E . ca!i colonics when
samples were plated from several separate small
volume cultures, as compared to samples tested from
a single large volume culture. Statistreally, this
indicated that mutations occurred randomly in the
separate small volume cultures, some early and some
late, resulting in the wide fiuctintfinri. In the large
volume cultures, fluctuations were within limits of
sampling error. However, the logic of this
experiment was not widely appreciated by
microbiologists, probably due to the complicated
statistical interpretation. It was the simple but
elegant'replica-plating^ technique ofLcderbctg and
1redeiberg (1952) that proved the point beyond ckuiht.
Using a velvet template, they were able to transfer
Lnooala from colonies on a master plate, onto a number
of other plates, retaining the relative positions iit the
colonies in all the plates. By such replica-plating on
culture plares with uhlI without b^tcriophage^ ditv
were able to show that bacteriophage resistant mutants
appeared without the bacteria ewer having had contact
with a selective agent.
Mutation may affect any gene and hence may
modify anv characteristic of the bacterium. Mutants
may vary in properties such as nutritional
requirements, biochemical! reaction, anti genic
structure, morphological features, colony form, drug
susceptibility', virulence and host range. The
practical importance of bacterial mutation lies
mainly in the field of drug resistance and the
development of live vaccines.
Transformation is the transfer
of genetic information through the agency of free
UNA. [t was the first example of genetic exchange
in bacteria to have been discovered, Griffith in 1928
found that mice died when injected with, a mixture
of live noncapsulated (R) pneumococci and heat
killed capsulatcd (S) pneumococci, neither of
wEheh separately proved fatal. If in the experiment,
the live (R) pneumococci wCK derived from
capsular type II and the killed (S) strain horn type
III, from blood cultures of the mice that had died,
live type 111 capsuktcd pneumococcus could be
isolated, showing that some factor in the heat killed
type 111 pneumococcus had transferred the
information tor Capsule synthesis to the live rough
strain. Such transformation was subsequently
demonstrated in vitro also. The nature of the
transforming principle was identified as DNA by
Avery, Macleod and McCarty in 1944.
The transfer of a portion of rhe
DNA from one bacterium to another by a
bacteriophage is known as transduction
Bacteriophages *re viruses that parasitise bacteria
and consist of a nucleic acid core and a protein
coat. During the assembly of bacteriophage progeny
inside infected bacteria, 'packaging errors' may occur
occasionally. A phage panicle may have at its cute,
iKisiJc^ iuown midtic athl, a segment ol die host
DNA. When this particle infects another bacterium,
DNA transfer is effected and the recipient cell
acquires new characteristics coded by the donor
DNA. Transduction may he ’generalised", when ir
involves anv segment of the donor DNA at random,
of it may be 'restricted', when a specific
bacteriophage transduces only a particular generic
trait. Restricted transduction has been studied
intensivebr in. the 'lambda' phage of E. gciJj. The
C o p y rig h te d m aterial
56 * Te>:lDook
p ro phage lam b d a lh Inserted in the b acte rial
chminoRomc nnlv between the genes- d ctcim in irg
galactose ulilLHalion (gu!'■ .md biotin synthem-i (bio)
and therelore it Iran-duces uolv
■f either o f these.
Transduction is not confined fo transfer of
chromosomal DNA, Epi-omcs and plasmids may
a I- ■ ■■ be transduced. IThe plasmids d e te rm in in g
penicillin resistance in staphylococci ,trc transferred
from cell to cell hy transductkin.
' fransductior appears to be the most widespread
mechanism of gene transfer among prokaryotes and
provides an excellent tool tor the gene'lic mapping
of bacteria. Any group of bactcda for which
bacteriophages exist can be subject to transduction.
It has been reported that transduction raiv
occasionally be effected in eukaryotic cells also.
Transduction has been proposed as a method of
genetic engineering in the treatment of some inborn
errors of metabolism.
L y s o g e n ic c o n v e r s io n : Bacteriophages
exhibit two types oflifecycle. In the virulent or Jj-laV
cycle, large numbers of progeny phages are built
up inside the host bacterium, which ruptures to
release them. In rhe tempera re or nonW c cycle .
the host bacterium is unharmed. The phage DNA
becomes integrated with the bacterial chromosome
as the prophage, which multiplies F^Tichronouslv
with the host DNA and is transferred to the
daughter cells. This process is exiled Lysogeny and
bacteria harbouiing prophages are called iysogenic
bacrena Lysogeny IS extremely frequent in nature.
In lysogenic bacteria, the prophage behaves as
an addi fional segment of the bacteri ;d chromosome.
Coding for new characteristics. Tin-. process by
which the prophage DNA confers genetic
information to a bacterium is called lysogenic or
phage connersfon. In transduction, the phage acts
only as a vehicle earn irlg baCteTixl genes imm one
cell to another but in lysogenic conversion the
phage DNA itself is the new genetic element.
Lysogenic conversion influences susceptibility to
bacteriophages (immunity to Rupctinfection with
o* Microbiology *
the same or related phages) and antigenic
characteristics, O f great medical importance is the
lysogenic conversion in diphtheria bacilli, which
acquire toxigcnicity {and therefore virulence) by
lysogcnisation with the phage beta. Elimination of
the phage from a toxigenic strain renders it
nonto\i genic.
f-o n ju R a tio n : Conjugation is the process
wherein a ‘male1 or 'donor' bacterium 'mates Ot
makes physical contact with a ‘female1or 'recipient'
bacterium and transfers genetic elements into irr
This has been considered to be the bacterial
equivalent of sexual mating in higher organisms
bur rhe analogy is irrelevant xs, following
conjugation, the female bacterium is in turn
converted into a male cell! Bxcten.ul conjugation
was first described by Lederherg and 1 :itum (1946)
ir a stml n of E, coYs called K12 and has been most
extensively studied in this strain.
Conjugation takes place between a male cell and
x female cell. T h e malcnc&s or donor status of a
cell Is determined by the presence in it of a plasmid
which codes for speci.iiised fimbria (sex pilus)
which projects from the surface of the cell. The
plasm ill DNA replicates and a copy of ir passes
from the donor to the recipient cell, probably along
the sex pilus (cwyqgation tube). As a result, the
recipient attains donor status and can in turn
conjugate with other female cells. The malencss in
bacteria is thus a transmissible or 'infections’
characteristic. Along with the plasmul DN A.
portions of the host DNA also are sometimes
transferred to the recipient. Tire donor DNA then
combines with the DNA of the recipient, effecting
genetic recombination. It wat in E. coli Kl2 that
the role of plasmids in cucijugxtion was first
recognised. The plasm id responsible was termed
the 'sex factor1or 'fort'.liry fh") factor'. When other
similar plasmids were also discovered, the term
'transfer factor came to be used lor .ill such plasm ids
which conferred on their host cells the ability to
act as donors in conjugation.
C o p y rig h te d m aterial
 The F factor is a transfer factor
Eilat contains the genetic information necessary for
the synthesis of the sex piius and for self-transfer
hut is -devoid of other identifiable genetic matkcta
such as drug resistance. Cells carrying rhe F factor
(F*cells) have no distinguishing features other than
their ability to mate with F~ cells and render them
F\ The F factor is actually an episome and has the
ahility t» exist in some celts in the 'integrated state’
or inserted into the host chromosome. Such cells
arc able to transfer chromosomal genes to recip ient
ce lls w ith h ig h freq u en cy and. are an own as \ Itr
cells. Following conjugation with an I Ift cell, an
V only rarely becomes F*„ though it receives
chromosomal genes from the donor.
This conversion of an V* cell into the Hfr state
is reversible, When the F factor reverts from the
integrated scare to die free scare, it uiay sometimes
cany with at some chromosomal genes from near
its site uf attachment. Such an F factor
incorporating some chromosomal genes IS called
an F prime (FO factor. When an P cell mates with
a recipient , it transfers, along with the F factor, the
host: genes incorporated with it. This process of
transfer of host genes through the F' factor
resembles transduction and has therefore been
called sr.vrjcjc-ijon [Fig. 8.3).
Several strains
of coliform bacteria produce colicms— antibiotic-
Lilte Hubstances which are specifically and selectively
lethal to other enterobacteria. As similar substances
arc produced by bacteria ocher than coliforms also
[pytHilo by Pjteidw(Vra-i pyocjn*it&ir dipliiherikin
by Corynebactcrium d ip h th e ria c), the name
bacteiiocin has beer given to this group of
substances. The specificity of action of bacteriocins
enables intraspccics classification of certain bacteria
(for example, Shigelh sormei, Ps. aeiugftiosa).
Colic in production is determined by a plasmid
called the Cot factor, which resembles the F factor
in promoting conjugation, leading to self-transfer
and, at times, transfer of chromosomal segments.
This
plasmid is of great medical importance as it leads
hj the spread of multiple drug resistance among
bacteria.
This extrachromosomal mechanism o f drug
resistance was first reported by Japanese workers
( 1 9 5 9 ) investigating the sudden in cre a s e in
infections caused by the Shigjetla strains, resistant
simultaneously to sulphonamides, streptomycin,
chloramphenicol arid tetracycline. They observed
that patients excreting such Shigella strains also
shed in their feces E. coli strains resistant to the
same drugs. Transfer of multiple drug resistance
was demonstrated between E. it)ti and Shigella
strains both in vitro and in vivo. The resistance is
plasmid mediated and is transferred by conjugation.
This mechanism of drug resistance is known as
trxns&mhk, episomsl or infection? drug1resistance.
This plasmid consists of two components - the
transfer factor called the resistance transfer fector
(R T F) which is responsible for eonjugataonal
transfer, and a resistance determinant (r) for each
of rhe several drugs. The whole plasmid (RTF + r
determinants) is known as the R factor. An R factor
can have several r determinants! and resistance to
as many as eight or more drugs can be transferred
simultaneously (Fig. 8.4). Sometime* die RTF may
dissociate from the r determinants, the two
components existing as separate plasmids. In such
cases, though the host tell remains drug resistant,
the resistance is not transferable. The RTF can have
attached to it determinants other than those for
drug resistance, Enterotuxin and hemolysin
production in some cPteropatllOgenilc E. coli are
transmitted by this transfer factor.
Transferable drug resistance is seen widely in
various pathogenic and commensal bacteria of man
and animals, such as Enterobacteria rate. Vibrio,
Pseudomonas., PasreureJ/a, The transfer can be
elfocted readily in vitro- but in the normal gut, it is
inhibited by several factors such as anaerobic
conditions, bile salts, alkaline pH and the abundattce
of anaerobic Gram positive bacteria minimising the
C o p y rig h te d m aterial
 liACTERJAL GENETICS
chances of contact between donor cells and suitable
recipient cells, But in the intestines of persons on
oral antibiotic therapy, transfer occurs readily due
to the destruction of the sensitive normal flora and
the selection pressure produced by the drug.
Transferable drug resistance is miw universal
in distribution and involves aLJ antibiotics in
common use. Its incidence is directly proportional
to the frequency of use of antibiotics in rhe area.
Bacteria carrying H factors can he transmitted from
animals to mart. Hence indiscriminate use of
antibiotics in Veterinary practice or in animal feeds
can also Lead to an increase of multiple drug
resistance in the community. The addition of
antibiotics in animal feeds has for this reason been
prohibited by legislation in some countries.
Widespread resistance has considerably diminished
the clinical efficacy of muse antibiotics.
1£ |E .f. i.■i.s Ml. B C H A rf I £■M § iJ . f* .IL?. jJ
'■SjL’lji / ,i".l ,i •-£ li H. Jo.%: i K
Bacteria fluty acquire drug renisUdfc bv mutation
or by one of the methods of genetic trsnufcr, The
biochemical mechanisms of i c^istance may be
several,, including decreased permeability to the drug,
develop me nt of altcmativr mctabolic pathways, a ltd
production of cittymes inactivating the cirug^.
Mutational resistance is mainly of two types:
0 ) the Htepwiae mut^don, as *een with penicillin,
where high levels of resistance arc achieved only
by a series of small-step mutinous; and (2) the
ll>he-step' mutation, as seen with streptomycin,
where the mutants differ widely in the degree of
resistance, some exhibitini; low resist a nee, while
others may be highly resistant; and some even
streptomycin dependent.
C o p y rig h te d m aterial
 In clinical practice, mutational resistance is of
great importance in tuberculosis. If a patient is
treated with streptomycin alone, initially the bid Hi
die in large numbers but soon resistant mutants
appear and multiply unchecked, IF two or more
antituberculous drugs are used Jut combined
treatment, rcpopulafion by resistant mutants does
not occur, as a mutant resistant to one drug will be
destroyed by the other drag- The possibility of a
mutant exhibiting re sis ranee to multiple drags
simultaneously it so remote as to be virtually
nonexistent. This is lIlv rationale behind combined
treatiilent in tuhaeuletin- 1 lowever, inspire of thi&
knowledge, inadequate or inappropriate treatment
over the years has caused extensive resistance in
tubercle bacilli, leading to a pandemic of multidrug
resistant tuberculosis (M D RTB) across lIil world.
Resistance transfer bv transformation can be
deirtoiwWaKd expe-rirncntaltv but its significance in
nature is not known. Acquisition of resistance by
transduction is common in ^Tiphydococcl. The
MATING
penicillinase plasmids, which are transmitted by
transduction, may also carry determinants for
resistance to mercuric chloride and erythromycin.
Transferable drug resistance mediated by the R
factor is the most important method of drug
resistance. Acquisition of an R factor simultaneously
confers resistance to several drugs and therefore
treatment with a combination of drugs is rot useful.
The resistance Is due to the production of degrading
enzyme-, ;und the Ieve I of res istance is UdUdly 11igli ■
Resistance maybe transferred between bacteria of
different monomicgroups. While resistant mutants
usually have a lower growth rare and reduced
virulence as compared Lo tile hvild strains, bacteria
earring R factor? are apparently normal in other
respects. K factors in some cases may even lead ru
enhanced virulence. Multiple drug resistance was
initially seen in bacteria causing diarrhea and such
olIll't mild infections that did not call for antibiotic
treatment as a routine. Bat subsequently li has
spread to virtually all pathogenir baL-tenn affecting
humans and amrnals, making antibiotic therapy ol
infections ineffective.
In the laboratory, R factors may sometimes be
eliminated by treating bacteria with acridine dyes
□r etliidium bromide. But in rhe communin', the
qrjy wuv to prevent widespread dissemination ul
multiple resistance is to restrict the use ofidtibifitbi
ro the essential minimum.
Certain structurally and genetically discrete
segments of DNA have been identified that have
the ability to move around in 1 CUt-and-plSte'
manner between chromosomal and extrach™-
mosomal DNA molecules within cells, These DNA
molecules ire called rrjosjjoxins {jumping genes’)
and this ruhie of genetic transfer, mujiipcsiriu-rt.Thr
earliest of such mobile genes was drsCovered by
Harhara McClintOck in plants during Work in the
1940s and 50s, rbr which she was awarded the
Nobel Pri^e for Medicine in 1983. A transposes is
a segment of DNA with one or more genes in the
centre, and the two ends carrying 'inverted repeat'
sequences oi nucleotidej — nucleotide sequences
complementary to each oilier but in the reverse
order. Because of this feature, each strand of the
transposon can form a single-stranded loop carrying
the gene, and a double-stranded stem formed by
hydrogen bonding Iselwccn the terminal inverted
repeat sequences (Fig. 8.51. Small transposons
(1-2 kb) arc known an 'insertion sequences' or IS.
One drug mixture at a time
Iam degree resistance
Dan overcome by hieh drug dose
Drug Lumbinations can prevent
Resistance -does nor spread
Mutants mar be defective
Virulence may be 1cm
Tra nsposons attach at certain regions of
chromosomal, plasmid or phage DNA. Insertion
of a transposon leads ro the acquisition of new
characteristics by the recipient DNA molecule.
Unlike plasm id?, nunsposons are not self replicating
and depend or, chromosomal or plasmid DNA for
replication.
hv transposition, a segment of the DNA can be
transferred from a molecule to another molecule
that h.L- nu genetic homology with either the
transposnblc clement or with fhc donor DNA. Jn
til is it differs from re-corn hi nation. As siieiible
chunks of DNA arc added by transposition, the
recipient irtolecule becomes heavier.
Characteristics transferred by transposons may
sometimes confer survival advantage under appropriate
environmental dominions, Ii has been suggested that
the reliance-determinant segments of the R factors
nny li.LVt evolved as collections of teanspusons, each
carrying a gene thar confers resistance to one CrTseveral
antibiotics.
Transposition is a mechanism for amplifying
genetic transfers in nature and has been identified
in microorganisms, plants and animals, Transposons
appear to accomplish in nature, gene manipulations
■similar to the laboratory' manipulations that have
been called 'genetic engineering1.
m m , m m m m
Discoveries in microbial genetics have provided the
basis for the discipline of molecular genetics, which
Multiple drug resistance
High degree resistance
Ftkb dose ineffective
Combinations cannot prevent
Spreads to same or different species
Not defective
Virulence not decreased
C o p y rig h te d m aterial
is concerned with the analysis anti manipulahon of
D N A using biochemical and m ierobiotogial
techniques. It lias Ih-lvi staled that these Techniques
have revolutionised the study of biology and
medicine* probably more than any technique since
the development of tilt light microscopes. Some
techniques and applications of molecular genetics
arc dibcushc-J below.
The most important
application of molecular genetics in biotechnology
is genetic engineering or recombinant DNA
(rDNA) technology. This conniHls of isolation of
the genes ending f'nr any desired protein from
microorganism* or from cells of higher forms of
hie Liieluding human beings, and their introduction
into suitable microorganism^!* in tvhich [he genes
would be functionaln directing the production of
the >peeifie protein. Such cloning of genes In
microorganisms enables the preparation of the
©
desired protein in pUM form, in large quantities
and at a reasonable Cost-
Different strategics have been employed for
obtain ing the desi red genes. In r very small protci ns,
such as the pituitary hormone sonaatostitin whose
complete ainnmacid HequeTifes arc known, the genes
can be synthesised in the laboratory. With larger
proteins* this is not possible. The DNA can be
cleaved by speciiic enzymes called restriction
endonucleases and the fragments containing the
desired genes isolated. This Joes not work with
DNA of higher organisms as they contain introns.
In such eases, flu- messenger KNA concerned car
be isolated from cells producing the desired protein,
A DNA Copy is made from the toRNA lining the
enzyme reverse transcriptase. 'Die diiublE-StMdcd
DNA gene is [ben prepared using DNA polymerase.
This is incorporated into suitable vectors or carriers*
such as, plasmids or temperate fracrcriophsgcs, for
62 < T e x t b o o k or M i c r o b i o lo g y »
in sertio n in to microorganisms. The microorganism
commonly employed is E, coli K l2, though many
other bacteria and yeasts have also been used.
Genetic engineering has become an established
branch of biotechnology with great scope for
commercial exploitation. Cloned human insulin,
interferons, somatostatin, gfuwth hormones and
many other biological! have already been marketed.
Safer vaccines can be produced by cloning the
protective antigens of pathogens,as has already been
done, as in the case of foot and mouth disease, and
hepatitis B and rabies viruses. This versatile
technique has manv extramedicaJ applications also.
R e s tr ic tio n eiH laim v L easts: [restriction
enzymes) are microbial enzymes which cleave
double-stranded DNA at specific oligonucleotide
sequences. Many such enzymes which act at
di tTeient nucleotide sequences (for example, Ecu
RI, H ind III, Taq I) have been recognised. The
natural function of restriction enzymes in bacteria
may be the destruction of foreign DNA that may
enter the bacterial cell
Restriction enzymes split DNA strands into
fragment^ of varying lengths. These can be
separated by gel electrophoresis and stained with
ethydium bromide and photographed.
I )N.\ probes: The specificity of the Interaction
in base pairing during DNA or RNA synthesis
enables the production of specific DNA probes.
These site radioactive, biotinylated or otherwise
labelled copies of cloned single-stranded DNA
fragments, usually 2 0 -2 5 nucleotides long and
containing unique nuclentitle sequencer which can
be used (hr the detection of homologous DNA by
hybridisation. DNA probes are being used
increasingly in the diagnosis of infectious diseases.
Probes contai ning sequences un ique to the microbc
(5trvi in, species or group} to be detected can be added
to microbial cultures, body fluids, tissues or other
materials suspected to contain the microbe nr its
DNA, The D N A probe hybridises with the
complementary spce^ic sequences on the microbe’s
DNA. The advantages of DNA probes for diagnosis
arc their high degree of specificity, ability to detect
minute quantities of complementary DNA even in
the presence of other microbes, and the capacity to
recognise microbes that arc either ditficult or
impossible to culture- DNA probes for the detection
of many pathogens are now commercially available.
Blotting techniques DNA fragments obtained
by restriction enzyme digestion and separation on
gel can be transferred from the gel by blotting to
nitrocellulose or nylon membranes that bind the
DNA. The DNA bound to the membrane is
denatured (convened to the single-scranded form)
and treated with radioactive single-stranded DNA
probes- These will hybridise with homologous
DNA To form radioactive double-stranded segments,
which can be detected on X-ray film. This highly
sensitive technique for identifying DNA fragments
by DNA : DNA hybridisation is called Sourhcm
blotting, after EM Southern who devised it. This
technique has very wide application in DNA
analysis.
An analogous procedure for the analysis of
RNA has been called northern blotting ( is opposed
to southern blottingE). f lere the RNA mixture is
separated by gel electrophoresis, blotted and
identified using labelled DNA or RNA probe;.
A similar technique for the identification of
proteins (antigens) is called immunoblorting [nr,
in conformLty with other blotting techniques,
western blotting). Here the protein antigen mixture
is separated by SD S-PA G E (sodium dodecyl
sulfate—polyacrylamide gel electrophoresis), blotted
on to nitrocellulose strips and identified by
radiolabelled or enzyme-labelled antibodies as
probes. The western blot test has received wide
publicity as the confirmatory'test for the diagnosis
of HIV antibody in sera. The specific] Ly of the test
depends on its ability to separately identify
antibodies directed against different antigens of the
pathogen (for example, against the surface, core
and reverse transcriptase antigens of HIV).
P o ly m e ra se ch a in re a c tio n ( P C R ) : This
is a rapid automated method for the amplification
 * Bacterial G enetics ► 63

of specific DNA sequences {or genes), invented by
Kary B Muliit in J9B3, far which he wtin the Nnhd
Prizv i:i Chemistry in 1993. PL Rconi-istE of several
cycles nf sequential DNA replication where rhe
products of the first cycle become the template for
the next cycle. Itmakes available abundant quantities
of specific DNA sequences starting from sources
containing minimal quantities of the same.
The technique is as follow*: two oligjonucleutide
primers complementary to the flanking region of the
DNA sequence to be amplified am incubated with
the target DNA, nucleoTides and DNA polymerase.
The reaction consists of three essential steps:
1. beat demtuiat ion of the sample DNA to si ni^ll-
strand;
2. annealing of sequence-specific oligonucleotide
priliners Co the boundaries nfthe DNA segment;
and
3. Extension of the primers by DNA polymerase
to form new double-stranded DNA across the
segment by sequential addition of
deoxynucleutides,
These three steps constitute one cycle of the
merlon. These cycles arc repeated several times,
usually for 20-50 cycles in the thermocycler, at the
end of whu'h hundreds of thousands of copies of
the original target sequences are available. As the
reaction steps take place at high temperature [50­
95 a heat-stable polymerase, such as Taq 1 has
to be employed.
With its enormous capacity to .Liuplitv DNA,
PCR is a versatile tool useful in diverse areas such
as diagnosis, of infectious, genetic or neoplastic
diseases, in forensic investigations, l:i
archeobiological studies of ancient specimens
and in the cx.loi ination oi phylugertet ic relutiurtshi[i'-
in evolution.
Based on the principle of PCR, other target
amplification systems have been developed. One
-■uch.. Transcription mediated amplification i 1 MAj
which amplifies ribosomal RNA instead of DNA
has been applied as a rapid diagnostic technique
for infections such as tuberculosis where cultures
are difficult or delayed.
M oleflu k r epidem iology: One offshoot of
molecular genetics is molecular epidemiology. Here
molecular methods such as ]i Iasmid ji nit! Lc an .lIvsi s,
genomic fingerprinting and PCR are used for the
identification and matching of microbial iMjlatcs
for epidemiological purposes.
G e n e tic m eppinjf: As a result of the
remarkable advances in molecular genetics, it has
been possible to delineate the complete genomic
sequences of bacteriophages and other viruses,
bacteria and iheir plasmids, and even of some
eukaryotes including minim.lIh. Quite apart from
the useful information it has provided in
microbiology, its success emboldened the
iutcmational svient ific commun ity to venture on the
"human genome project7, the most expensive and
ambitious scientific project SO far undertaken in
biology. The results of this mammoth study became
available by the dawn of the twenty-first century
and have opened vistas in human biology and
medicine, as well as controversies and dilemmas
that Transcend medicine.

Further Reading
Hardy K. 19fJ6. Bacterid! Plasmidr- 2^ cdn. Rsinhold.
Innia MA rt J, 1990. PC’J? /Yutocok, San Dkro; Academic l1™ -
MulLin KH. 1vv ' ' ^lii- |'ii.\-iv.iT.i'i‘ /h.iin rf.ii lion. Nnhel Lecture, Stockholm.
Sambroak J er aL 19#9. Xlotccuhr Cloning. Cold Spring Harbour Laboratory
Tompki he LS. 1992. The use of molecular method* in infactioiiE diseases, .Vw EnglJ Med, 327:1290.
Towner KJ and A Cockayne 1993. Molecular Methods forMicrobial AJrnlifrcati-jn and Typing. London: Chapman-Hall.
W-t’.iui JD et al. 19tf3. Recomb,ii.hir DIVA. New York: Scientific American Books.

C o p y rig h te d m aterial
 Infection
Infection ind immunity involve interaction
between the annual body (host) and the infecting
microorganism. Based on their relationship to
their hosts, m icroorganism s can be classified as
saprophytes (from Creek sapros decayed; and
phvTilit plant) ami patiHircs. S.-iprophyto are free-
living microbes that subsist on dead or decaying
organic matter. They arc found in soil and water
and play an important role in the degradation of
organic materials in nature. They arc generally
incapable of multiplying on living tissues and
therefore are of little relevance in infectious
disease. Exceptionally, however, some saprophytes
like B. iubalin may infect devitalised hosts whose
natural resistance « greatty reduced (opportunistic
infection). Parasites arc microbes that can establish
themselves and multiply in hosts. Parasitic
microbes may be either pathogens (Hum Greek
pathos1suffering, and gen produce, that is, disease-
producing) or conunensais (from Latin com with;
and metrat, table, i.e., Living together). Pathogens
are mic roorgan isms that arc capable of produc ing
disease in the host. Commensal microbes live in
Complete hjti cicir1.^ with the host without eausi ng
any damage to it. The normal bacterial flora of
the body consist largely of commensals. Many
commensals behave as facultative pathogens in
that they can produce disease when the bust
res i-lance is Inwened-
It is necessary to di sringu i-ih between the term
‘infection and "infectious diseaseThe lodgement
and multiplication of a parasite in or on the tissues
of a host constitute infection. It docs not invariably
result m disease. In fact, disease is but a rare
consequence of infection, which is a common
natural event.
Infections may be classified in various ways.
Initial infection with a parasite mu host :s termed
primary' infection. Subsequent infections by the
same parasite m the host are termed re.mfecrjons.
When a new parasite sets up an infection in a
host whose resistance IS lowered hy a preexisting
infectious disease, this is termed secondary
infection. The term fo o t! infection (more
appropriately focal sepsis) indicates a condition
where, due to infection or sepsis ar localised sites -
such as appendix or tonsils, generalised effects
are produced. When in a pai lent already suffer] ng
from a disease a new infection is set up from
another host or another external source, it is
termed cross-infection. Cross-infections occurring
in h<Kpitd s are CfllIed : hwoa >:iii.i! sided inn* (tiKtr:i
Greek nosoconuon hospital}. The term iatrogenic
jrtfecfjun refers co phvsician-induced infections
resulting from investigative, therapeutic or other
procedures. Depending on whether rhe source of
infection is from the host's own body or from
external sources, infections are classified as
cndc^enous or exogenous, respectively. Based on
the clinical effects of infections, they may be
clasi-shed into different varieties. Inapparent
infection Ls one where clinical effects are not
apparent. The term suhcJjmcjil in/ecbon is often
used as a synonym. Atypical infection is one in
which the typical or characteristic clinical
man itestations of the particular infectious disease
are not present. Some parasites, following
infection, may remain in the tissues in a latent or
C o p y rig h te d m aterial
 i mfsction
ini. Lilen form pro 11fern mg and producing cluneal
disease when rhe host resistance is lowered- This
is termed intent .'idccrran.
S o t iujhs o p I n f e c t io n
I Inm ans The commonest source ol infection
for humans are humans ihemselvcs The paraiite
may originate from a patient or a carrier. A carrier
is a person who harbours the pathogenic
microorganism without suffering from any ill-
effect because of it. Several types of carriers have
been identified. A healthy carrier is one who
harboui ■-the pathogen bait lias never suffered from
the disease caused by the pathogen, while a
convalescent carrier ih one who has recovered
from the disease and continues to harbour the
pathogen in his body. Depending on the duration
of carriage, carriers are classified as temporary
and chronic, I’hc temporary earner state lasts less
than six months, while chronic carriage may last
for several years and sometimes even for the rest
of one’s life, llie term contact carrier is applied
to a person who acquires the pathogen from a
patient, while the term paradoxical earner refers
to a carrier who acquires the pathogen from
another carrier.
A n il no Is: M a n y pathogens are able to infect
both human beings and animals. Animals may,
therefore, act as sources of human infection. In
some instances, the infection in animals, may be
asymptomatic. Such anim als serve to maintain the
parasite in nature and act as the reservoir of human
infections. T h e y .ire, therefore, called reservoir
hontf. Infectii -us diseases tramsrn Ltted from an itrials
to human beings are called zooncuer. Zoonotic
diseases may be bacterial (plague from rats), viral
{rabies I :oiti dogs), p ro to n d l (tOxrh| iii> irtti sis [ n m i
cats), helminthic (hydatid disease from dogs) or
fungal (wuplnjic dermatophytes from cats and
dogs).
In suets; Blood sucking insects may transmit
pathogens to human beings. The diseases so
t 65
caused are called arthivpod-borrie diseases. Insects
such as mosquitoes, licks, mites, flics, fleas and
lice that transmit infections are called vectors.
Transmission may be mechanical (for example,
transmission of dvscnrerv or typhoid "linill: hv
the demesne fly). Such vectors arc called
m echanical vecrors, In other instances, the
pathogen multiplies in the body of the vector, often
undergoing part oil its developmental cycle in ir.
Such vectors are termed biological vectors (for
example, Aedesitqgypri mosquito in yellow fever.
Anopheles mosquito in matana). Biological
vectors transmit infecriun only after the pathogen
has muiliplied in them sufficiently or has
undergone q developmental cycle- I'he intcrv:il
between the time of entry of the pudiogcn into
the vector and the vector becoming infective is
called the exmmic incubanoji pern iJ.
Besides acting as vectors, some insects may
also act as reservoir hosts (for example, ticks in
relapsing fever and spotted fever). Infection is
maintained in such insects by transovarial or
tiansstadnd passage.
Soil and w ater: Some pathogens can survive
in the soil for very long periods. Spores of tetanus
bacilli may remain liable in the soil for several
decades and serve as the source nfinfectionr Fungi
(Hntopiasmn capsuiatum, Nocardia astcroidcs)
and also parasites such as roundworm and
hookworm survive in the soil and cause human
infection.
W.Ui-T may act as the source at infection cither
due to contam ination with pathogenic
microorgan isms { cholera vibri u, infective hepai itis
virus) or due to the presence of aquatic vectors
{cydups in guineaworm infection).
Food: Contaminated food may act as a source
of infection. The presence of pathogens in food
may be due to external contamination (food
poL%oniiLg by staphylococcus) or due to pre­
existent infoctii ?n in meat or other am mal products
(salmonellosis).
C o p y rig h te d m aterial

Infection may be acquired by contact,
which may be direct or indirect. Seaually
transmitted diseases such a? syphilis and
gonorrhea illustrate spread by direct contact. The
term COrtQ^ious disease had been used for diseases
transmitted by direct contact, distinct from
mfeciions disease signifying all other modes of
transmission.This distinction is now not generally
employed. Indirect contact may be through the
agency of fomifes, which are inanimate objects
such as clothing, pencils or toys which mav be
contaminated by a pathogen from one person and
act as a vehicle for its transmission To another.
Pencils shared by school children may nice as
fbmites in the transmission of diphtheria., and face
towels in trachoma.
Respiratorv infections such as
influenza and tuberculosis are transmitted by
inhalation of the pathogen. Such microbes are
shed by the patients into the environment, in
secretions from the mtse or throat during sneezing,
speaking or coughing. Large drops of such
secretions fall to the ground and dry there.
Pathogens resistant to drying may remain viable
in the dust and act as sources of infection. Small
droplets, under 0.1 mm in diameter, evaporate
imnwdiarely to become minute particies or dro/Jcf
nuclei (usually 1—10 pm in diameter) which
remain suspended in rhe ah for long periods,
dieting as Sources ut infection.
Intestinal infections arc generally
acquired hy the ingestion of food Or drink
contaminated by pathogens. Infection transmitted
by ingestion may be w aterborne {cholera),
foodborne (food poisoning} or handborne
(dysentery). The importance of fingerborne
transmission is being increasingly recognised, not
only in the case of pathogens entering through
the mouth, bur also those that enter through the
nose and eyes.
Pathogens, in some instances, may
be inoculated directly into the tissues of the host.
Fctanue spores implanted in deep wounds, rabies
vmts deposited subcutaneously hy dog bite and
arbrjviruses injected by inscct vectors are HCatHples.
Infection by inoculation may be iatrogenic when
unsterile syringes and surgical equipment are
employed. Hepatitis ii and rhe Human
lm mu nodeficiency Virus (H IV ) may be
transmitted through transfusion of inhered blood,
or the use of contaminated syringes and needles,
particularly among addicts of injectable drugs.
Insects may act as mechanical or
biological vectors of infectious diseases,
Some pathogens are able to cross
the placental barrier and infect the fetus in Utcro,
Tins is known as verticstl transmission. This may
result in abortion, miscarriage or stillbirth. Live
mtints may be born with manifestations of a
disease, as in congenital syphilis. Intrauterine
infection with the rubella virus, especially in the
first trimester of pregnancy, may interfere with
organogenesis and Lead to congenital
malformation. Such infections are known as
teratogenic infections.
Infection may sometimes be transmitted during
administration of injeciions1 lumbar puncture and
Cltheterisatifia, it m eticulous cure in asepsis is
lacking. Modern methods of treatment such as
exchange transfusion, dialysis, and organ transplant
surgery have i ntreased the possibilities for iatrogenic
infections. Laboratory personnel handling infectious
material are at risk and special care should be taken
to prevent laboratory infection.
The outcome of an infection will depend on
the interaction between microbial factors which
predispose to pathogenicity and host factors which
contribute to resistance.
The terms 'pathogenicitv* and 'virulence* refer to
C o p y rig h te d m aterial
die Ability of a microbe to produce disease or tissue
injury but it is convenient to make a fine
distinction between them. 1567*91023Pathogenic! tv' is
generally employed to refer to the ability of a
microbial species rn produce disease, while the
term ‘virulence1 is applied to tile saute property
in a strain of microorganism. Thus the species
M. Rj/jen-ti/osTS or the polio virus is referred to
as being pathogenic. The pathogenic species
M. fubcA’uJasis and the polio virus contain strains
of varying degrees id virulence including those
which arc avirulcnt, such as the vaccine strains.
The virulence of a strain is not constant and may
undergo spontaneous or induced variation.
Enhancement of virulence is known as ejtaJfarjon
and can be demonstrated experimentally by serial
passage in susceptible hosts. Re-duct ion ot
virulence is known as am?n natron and can be
achieved hv passage through unfavourable hosts,
repeated cultures in artificial media, growth under
high temperature or in the presence of weak
anriseptics, desiccation, or prolonged storage in
culture.
Virulence ls the sum total of several
determinants, as detailed below.
The initial event in the pathogenesis
1. PfiPltipH
2. Heat labile
3. Actively secreted by ceLs; diftuse into
sunuuEKlLn.ir medium
4r Readily separable from cultures by physical
means fueh as filtration
5. Action often erm.miL
6. Specific pharmacological effect for each
exu toxin
7. Specific tissue atiftniriee
S. Active in very minute doses-
9. Highly antigenic
10. Action specifically neutralised by antibody
of many infections is the attachment of the bacteria
to body surfaces. This attachment is not a chance
event but a specific reaction between surface
receptors on host cells and adhesive structures
(ligands) on the surface of bacteria. These adhesive
structures are called adhesins. Adhesins may occur
us organised structures, such as. fimbriae or
fibrillar and pill, or as colonisation factors. This
specific adhesin may account for the tissue
troplsms and host specificity exhibited by many
pathogens. Adhenins serve as virulence factors,
and loss of adhesins often renders the strain
avirulenl. Adi resins are usually made of protein
and are antigenic in nature. Specific immunisation
with adhesi us has been attempted as a method of
prophylaxis in some infections, as for instance
against E. pair diarrhea in calve* and piglets, and
gonorrliea in human bcings.
Tbis refers to the ability of a
pathogen to spread in the host tissues after
establish!rtg infection. Highly invasive pathogens
characteristically produce spreading or generalised
lesions (c.g.„ streptococcal septicemia following
wound inlection), while Less invasive pathogens
cause more localised lesions (e.gr. staphylococcal
abscess). Some pathogens, though capable of
1 jprtprtlysaccharides
He-at stable
Form part of cell walk do not diffuse into
surround ing medium
Obtained only by cell Ivsh
No en^mic action
Effect nonspecific; action common to all
endotoxins
N o specific tissue affinity
Active only in very large dost*
Weakly antigenic
Neutralisation by antibody LncfieccLw
C o p y rig h te d m aterial
causing serious or even fa fa] diseases, lack
imnsivcdiew .Lkogetluer (e.g., the LctanUS bacillus
which remains confined to the site of entry and
produces the disease by elaborating a potent
toxin).
Hucteria pnidllCt two types of
toxins— exotoxins and endotoxins
Exotoxins are heal labile proteins which are
secreted by certain species of bacteria and diffuse
readily into tile £UfK>Uflding medium. T I k v \ifC
highly potent in minute amounts and constitute
some of the most poisonous substances known.
One mgofletaiuLH or botulinum toxin is sufficient
to kill more than one million guinea pigs and it
has been estimated that 3 kg of botulinum toxin
car kill :lE1 the inhabitants of the world. Treatment
of exotoxins with formaldehyde yields toxoids
which, arc nontenure but retain the ability to induce
antihodien {antitoxins).Thevexhibit s-j'H.'cifLCtissue
affinities and pharmacological activities, each
toxin producing a typical effect which Can be
tnade otii by characteristic clinical manifestations
or autopsy appearances. E xoD oeub are generally
formed bv Grain punitive bacteria hut may also
be produced by some Gram negative organisms
such as Shiga's dysentery bacillus, vibrio cholera
and enterotoxigenic E. coli.
Endotoxms arc heat stable lipopotysaccharidcs
{I.PS) which form an integral part of the cell
wall of Gram negative bacteria. Their toxicity
depends on the lipid component (lipid A). They
arc not secreted outside the bacterial cell and arc
released only by the disintegration of rhe eel1 wall.
They cannot be coxoided, They are poor antigens
and their toxicity is not completely neutralised by
the homologous antibodies. Tliey arc active only
in relatively targe doses. T h ey do nor exhibit
specific pharmacological activities. All endotoxins,
whether i ripluted from pathogenic or
nonpathogenic bacteria, produce similar effects.
Administration of small quantities of endotoxin
in susceptible animals causes an elevation ot body
temperature manifested within 15 minutes and
laming tor several hours. The pyrogenic effect of
fluids used for intravenous administration is
usually due to rhe presence of endotoxins from
contaminant bacteria. Intravenous injections of
large doses ot endotoxin and massive Gram
negative septicemias cause endotoxic shock
marked by fever, leucnpenia, thrombocytopenia,
significant till in blood pressure, circulatory
collapse and bloody diarrhea leading to death
(Tables 9.1, 9.2).
Genes coding for some virulence
characteristics may be plasmid borne, Examples
ol plasmid-borne virulence fsLctors are surface
itorigtns responsible for the colonisation of
intestinal mucosa by E. coli and enuercuoxin
production hy E , cn/i :md Sfjtp/i. aureus. Multiple
drug resistance (R) plasmids increase the severity
of clinical disease by their resistance to antibiotic
therapy.
The classical example of
phage directed virulence is seen in diphtheria. In
diphtheria bacilli, the gene for toxin production
is present in beta or Other Box" curyncpbages.
The ability' of a paras ire
to spread from one hosr to another is known as
oanimumobiliry.This property does not influence
the production of disease in an individual host
but determines [he survival and distribution of a.
parasite in a commnnity. A correlation reed not
exist between virulence and communicability. In
fact, a highly virulent parasite may nut exhibit a
high degree of communicability' due to its rapidly
Lethal effect on the host. In general, infections in
which the pathogen is shed in secretions, as in
respiratory or intestinal diseases, arc highly
Communicable, In some instances, as in
hydrophobia, human infection represents a dead
end, there being an interruption in the spread of
the pathogen to other hosts.
Development ol epidemic and pandemic
diseases requires the strain of pathogen to possess
high degrees of virulence and communicability.
Some bacterial
C o p y rig h te d m aterial
products other than Toxins, though devoid n£
intrinsic toxicity, may comribute to virulence by
Inhibiting the mechanisms of host resistance.
Pathogenic staphylococci produce a thrombi n-
likt enzyme coagulant: which prevents
phagocytosis by forming a fibrin barrier around
the bacteria and walling off the lesion.
Fibfinolysitie promote the spread of infect in>ns bv
breaking down [he fibrin barrier in [issues.
HyaJuronidases split hyaluronic add which is a
component of intercellular connective tissue and
thus facil irate the spread, of infection along tissue
spaces. LcucocidinE damage polymorphonuclear
leucucvtes. Many pathogens produce hemolysins
capable of destroying erythrocytes bui their
significance in pathogenicity is not clearly
understood.
CapsuLuted bacteria
such n* pneumococci, pneumoniae and / /
influcuzae arc not readily phagocytosed. Some
bacterid surface antigens such as fhe Vi antigen
of 5. tvphi, K antigen* of E , cro/i also help the
bacteria to withstand phagocytocis and the lytic
autiviiy of compLcment.
Successful infections require
[hat an adequate number o f bacteria should gam
entry into the host. The dosage may be estimated
as the minimum infecting dose (M ID I or
m in im u m Lethal dose (M L D ) w h ic h are,
respectively, the minimum number of bacteria
required to produce clinical evidence o f infection
or death, respectively, in a susceptible animal under
standard conditions. As animals exhibit
considerable individual variation in susceptibility,
these doses an- more correctly estimated as
statistical expressions. JD 50 and LD 50, as the
dose required to infect or kill 50 per cent of the
animals tested under standard conditions.
Some bacteria, such as
streptococci. Call initiate infection whatever be the
mode of entry. Others can survive and multiply
only when introduced bv the optimal routes.
Cholera vibrios are infective orally but are unable
to cause infection when introduced subcutaneously. jr
This difference is probably related to modes by
which different bacteria are able to initiate tissue
damage and establish lliecusehv-.. Bacteria also
diffet in their sites of election in the host body
after introduction into tissues. They also differ in
the ability' to produce damage of different organs
in different species of animals. Tubercle bacilli
injected into rabbits cause lesions mainly in the
kidney and infrequently in the liver and spleen,
but eil guinea pigs die lesions ate mainly in the
liver and spleen, the kidnevs being spared. The
reasons for such selective multiplication in tissues
are largely obscure, though they may be related,
to the presence in tissues of substances that may
scLeciivdy hinder or favour their multiplication,
Infectious diseases maybe localised or generalised,

fyrogenicity Lethal action Depression of blood pressure

Activation of complement
Leumytuiir
Miierophagt iiihibinnii
IntravHculai crmgvitiiTion
Inhibition of glucose and
glycogen synthesis in the liver
Interferon release
LcuCopcnia
Stimulation of R lymphocytes
Induction of pmsrigkcuiin
synthesis

(Clotting of limuius lysate (lysate of amebocytes from hone-shoe crab, Limulus polyphemus, used as a
M for ikicuiun of endotoxins). J

C o p y rig h te d m aterial
70 * Texibook of Microbiology *

L o c a t e d mfccfLons may be superficial or lJc;il|i depending on their spread in the community,

seated. Generalised infection involves the spread
of the infecting agent from the sice of entry by
contiguity, through tissue spaces or channels,
along the lymphatics or through the bloodstream.
Circulation of bacteria in the blood is known as
bacteremia. Transient bacteremia is a frequent
event even m healthy individuals; and may occur
during chewing, brushing of teeth or straining at
stools. The bacteria are immediately mopped up
by phagocytic cells and arc unable to initiate
infection, bacteremia ofgreater severity and longer
duration in seen during generalised infections as
in typhoid fever, Septicemia is the condition
where batten .1 rircula.te and multiply in the blood,
form toxw products and cause high, swinging type
of fever. Pyemia is a condition where pyogenic
bactena produce septicemia with multiple
abscesses 1 0 the 1oteroal organs such ,lh the rplce n,
liver and kidney.
infectious diseases may be classified into different
types. Endem ic diseases arc those which are
constandy present in a particular area. Typhoid
fewijcndcnii' in most parts of India. An epidemic
disease is one that spreads rapidly, involving many
persons in an area at the same time. Influenza
causes annual winter epidemics in the cold
countries. A pandemic is an cpideni if that spreads
thmugh many areas of the world involving very
Urge numbers of people within a short period.
Influenza, cholera, plague and cntcroviral
conjunctivitis are pandemic diseases. Epidemics
vary in the rapidity of spread. Ifl&tcrbcrpc di^vcascs
such as cholera and hepatitis may cause explosive
outbreaks, while diseases which spread by person-
to-person contact evolve more slowly. Such
creeping or smouldering epidemics, as that of
cerebrospinal fev«r, are termed pnosotJenuc
dfofttSCSr

F u r t h e r H c it J i 11 £
Mi:ri' C A 1?S7. The ilitifrcpienejj-, o f Infectious Disease. J*1 edn. London: Academic Pm*.
Rati i..k S and MJ Larkin 1995. /itiJrtvrtciAqpraf and AfoJbndar aspects ofBacrer'ol V'ifufenoe. Chic hearer: Wiley,
IVwtun lft and Jl] Aibuthnot 1990. Determinant* of bacterial vi ruleiwt In: Tr^ntfj^jn d WfJmn Prindpk-s o f Bacteriology,
VrfyhijQ' tn<j Imifivnity, 31h cdn. VbLI. London; Edward Arnold.
Roth JA et ai, rds 1989. VriWcncr jVfccliaj.,.:,imf o f Ptrhngen*. Washington DCr American Society for
Microbiology.

C o p y rig h te d m aterial
 Immunity

The term 'immunity* traditional])' refers to the
resistance exhibited by the host towards injury
caused by microorganisms and their products.
However protection against infetrinus diseases is
only one of the consequences of the immune
response, which in its entirety is concerned with
the reaction of the body against any foreign antigen.
Immunity against infectious diseases is of
deferent types:
I Innate (Native) Immunity
general, or specific where resistance to a particular
pathogen is concerned.
Innate immunity may be considered at the level
of the species, nice or individual. Specie immunity
refers ri> thi.- total or relative refractoriness tu a
pathogen, shown by all members of ,i species. For
instance, all human beings are totally unsusceptible
to plant pathogens and to many animal pathogens
such as rinderpest or distemper This immunity is
something a person obtains by virtue of being a
part of the human species. The median isms of

E
Species species immunity arc not clearly understood but
Racial may he due rn physiological and biochemical
Individual differences between rfic tissues of the different host
sped cs, whi L-hdetermine wI ml he r or not a pat iiojnt;n

E
Species can multiply in them. An early insight into the basis
Racial nf species immunity was game d by Pasteui’s
Jndividual experiments an anthmx in Iro^s, which ate natuially
resistant to the disease but become susceptible when
IT Acquired (Adaptive) Immunity their body temperature is raised from 25 “C to
Natural 35 *C,
(a) Active Within a species, different races ir:.u- show
Artificial differences in susceptibility to infections. Tliis is
known as ■ .. '..■■Ii n -i ■■ ■■!
Natural h ■ : : i ! : I1 . : rr '-n-t r ■ ■ d \ e rm i i li t r y n ■
(b) Plassive mi t'hr.\>, Such racial differences arc known to be
Artificial genetic in origin, and by selection and inbreeding,
it is possible to develop, at will, races that possess
lnr:jtc or native irmnuru.'i ^ rtu resist a nee to high degrees of resistance or susceptibility to
infections w hich an individual poisc^sc*; bv virtue various pathogensL It is difficult to demonstrate
t hi? i^cnefi .uul i onstitutii nu.1 inak up It is not marked differences in immunity in human races, as
affected by prior contact with microorganisms or controlled breeding is not possible in the human
immunisation. It may be nonspecific when it species, ir hie been reported that the people af
indicates a degree of resistance to infections in Negroid origin in die USA aic mom susceptible

C o p y rig h te d m aterial
72 * Tpiiibaok pf Microbiology x
than ihe Caucasians to tuberculosis. But such
comparkcms are vitiated by external influences such
as differences in socioeconomic levels, An
interesting instance of genetic resistance to
Plasmodium hkipam m malaria is *cll: i m some
parts of Africa and the Mediterranean coast A
c:r<-l3Lr;iry abnormality of red cells (sickling},
prevalent in rhv area, confers immunity to infection
by the rn.LlLiri.Ll parasite and may have evolved from
the survival advantage conferred by it in a malard
environment.
To: v I : ■.■.■ i: i I : ■'■ i i ■.' n"'- l' '
i ■I ' i ... i* ihiIl’/ uIlu : i ■ know . as
indimhii} inwiuoir\ The genetic basis of individual
immunity in evident finm studies on the incidence
of infectious diseases in twins, Ir is well documented
that homozygous twins exhibit similar degrees of
resistance or susceptibility to lepromatous leprosy
and tuberculosih. Such correlation if not seen ill
heterozygous twins.
Several factors influence the level of innate
immunity in an individual:
Afce: The two extremes of life carry higher
susceptibility to infectious lLikc.lhc!; as compared
with adults. Tbe fetus in mcro is normally protected
from maternal infection by the placental barrier.
But some pathogens cross tins barrier causing
overwhelming infection;; resulting in tctal death-
Smnc, such as rubella, hetpeshcytomegaloviruses
and ToiopJa^rtfa gemefu, lead to to r genital
malformations. 1 he heightened susceptibility of the
fetus to infccti...... s related to the immaturity of its
immune apparatus Newborn animals are mme
susceptible to experimental infections than older
ones. CossacknLviruses cause fatal infection in
suckling mice but not in adults.
Increased susceptibility in the young msy, in some
instances, be due to hormonal influence. Tinea capitis
C^UEed by A h I. .. Iri
undergoes spontaneous cure with the onset oi puberty.
The susceptibility nl the vaginal epithelium in
prepubertal girls to gonococcal infection is another
nsMno: H>t tlu effect ■ : h*i: 'ine* i resistance
Some m lectioii'. Ill jxilimiiv: I it i'- and
chK'ken|sox lend In he more seven ir. iduits than
in vi>urig children, due to hypersensitivity rhai
causes g m le i tissue damage. Conversely, hepatitis
H i - : i ■I . .^ -s I-■=■il i : ■ 11■■ i
Asymptomatic because clinical disease requites
i rtm'.'.mc res pi winch is lac kin it th.it
•L^e. Hovrever, the virus multiplies unrestrained and
such neonates end up as chronic viral carriers, often
developing late hepatic complications. Old persons
arc highly susceptible to infections due to the
waning of their immune responses and other
infirmirics like enlarged prostate leading to urinary
stasis.
H orm onal influences: Endocrine disorders
such as diabetes mellirus, hypothyroidism and
adrenal dysfunction arc associated with an enhanced
susceptibility to infections. The high incidence of
staphylococcal sepsis in diahetes maybe related to
the increased level of carbohydrates in tissues,
i 'ortK. i-'. rou!- c uti an impurtant ir tl icncc mi t u
response to infection. They depicts the h o s t 1:
re<i ’.r ,i net* h\ i •:. : i 11 ' 111 *1a •i i i i :■■• and
.inriphago,\tii. c flo rs ind h\ lit - iip| u - -on of
antih: :i ind hvprisenauivitx ! m lIm
have a beneficial effect in that they neutralise tlie
harmful cficct of bacterial products such ns
endotoxins. The elevated steroid level during
p n .g r u n o ma\ he tc ited to the heightened
susceptibility of pregnant women ti> itianv
inkvtii 'ii' The reported effect of stress in increasing
susceptibility to infect ions, may in some measure
be due tn the release of stcroids-
Nutrithffl: The infraction between malnutrition
and immunity is complex but, in general, both
humoral and cell mediated immune processes are
reduced when there is malnutrit ion. Cell mediated
immune responses such as the Mantoux test become
:ii ■
■ negative in severe ptotein deficiency, as in
kwashiorkor. Because of its wide prevalence,
malnutrition may well be the commonest cause of
immunodeficiency.
P.Ltjd:ixlcjlIv, there is some evidence that certain
C o p y rig h te d m aterial
Infections may nor become d i n i apparent in
the severely malfiuufished. Malarial infection in
rhe famine stricken may not induce fever but once
clteir nutrition is improved, clinical malaria
develop. It ban also been reported that some viruses
may not multiply in the tissues of severely
malnourished individuals.
M e c h a n is m s o ? I n n a t e I m m u n it y
I he in ti, i '■km and
to h' iv.v :m-ir.!'r: i ■ covering the hod)' protect if
considerably against invasion by microorganisms,
They provide much more than a mechanical
barrier, i h J t h '-km i' d k ^ ' h.u kinuM i . umu
(o w hich tltc presence (if ln^:i concentration ol -,i!t
ii 1 1 <• iir\'ni_ \ur.u, flu- scKk I'tHi'i "v. retiuriK and
tin- lt>n_ ,l.i n t.!in I. uh and . ; , mcrihute.
v i i h
When cultures of typhoid bacilli placed on healthy
skin and on a glass surface are sampled at intervals,,
the bacteria cm the skin are seen to he killed within
minutes, while those on glass survive for several
houm. The bactericidal activity of skin secretions
is illustrated by the fittjueni mycotic and pyogenic
infections seen in persons who immerse their hands
in soapy water for long periods occupationally.
Though the skin frees itself readily of bacteria
deposited on if (transients), its reactions are different
to the bacterial flora normally resident on it-
kesident flora are ro t easily removed even hy
washing and application of disinfectants-
I I . imiui-.i iC rlii' rr-.pir.imi\ tract has several
him re mechanisms of defence. The very architecture
of" the nose prevents entry of microorganisms to a
large extent, the inhaled particles being arrested, at
or near the nasal orifices. Those that pass beyond
arc held by the mucus lining rhe epithelium, arid
arc swept back to the pharynx where they tend to
he swallowed or coughed GUL The cough reflex lS
an important defence mechanism of the respiratory
tract. The cilia on the respiratory epithelial cells
propel particles upwards. Masai and respiratory
secretions contain mucopolysaccharides capable of
combining with influenza and certain other viruses.
Panicles that manage to reach the pulmonary alveoli
are ingested by the phagocytic cells, present there.
The mouth is constantly bathed in saliva which
has an inhibitory effect on many [mcroorgarnsms-
Parridcs deposited in the mouth are swallowed and
subjected to the action of the digestive juice*. Tile
high acidity of the stomach destroys most
microorganisms, The pll becomes progressively
alkaline from the duodenum ro the ileum. The ileum
contains a rich and varied flora and in. the large
intestine, the bulk of rhe contents is composed of
bacteria.
The intestinal mucosa is Covered by a lacelike
network of mucus. Particles get enmeshed in the
mucus and! form small innisei which are propelled
by peristalsis.
The conjunctiva is freed of foreign particles hy
rhe flushing action of lachrymal secretions. The
eyes become susceptible to infection when
lachrymal secretions are absent. Tears contain the
antibacterial substance lysozyme, first described hy
Fleming {1^ 22). This is a thermolabile low
molecular weight basic pro rein which acts as a
muraminidase. I v - :v- i- r -'.nr in m-m- IInh.i-
a m i 11 ■i l‘;. i . ■* i.l sa v i e t i n tv- m... v p r •I’ v b r j i
rlii .1, ■■.'■ I 'i .ml- It acts by splitting certain
polvsaccharide components of the cell walls of
susceptible bacteria. In the concentrations seen in
rears and other secretions, lysozyme is active onlv
against some nonpathogenlc d ram positive
bacteria However, it occurs in phagocytic ceils in
concentrations high enough to be lethal to many
pathogens.
The flushing action of urine eliminates bacteria
from the urethra.''pi i in ii ■ i d - - . . mi the
sem en carry out antiliacteriLiI acip, Ll \ TTie acidity
of the adult vagina, due to the fermentation of
glycogen in the epithelial cells hy the resident
aciduric bacilli, makes it inhospitable to many
pathogens.
Aiitibftts'teri&i subtle, ?:.i. 'isloctd
"I he complement system pOstfciSCS
bactericidal activity1 and plays an important role in
C o p y rig h te d m aterial
74 * Textbook ai Microbiology >
the desmani>n of pathogenic bacteria that invade
the blood and Tissues (see chapter 14),
Several substances possessing antibacterial
properties have been described in blood and tissues.
These include (1 } beta lysm, a relatively
thcrroM-cable substance active against anthrax and
related bacilli; (2) basic polypeptides such as
Lukins extracted from leucocytes and plakins from
platelets, i ■iI acidic substances, such as lac lie acid
found in muscle Tissue and in the inflammatory
Tores; And (4) lactnperosddase in milk. While these
substances possess antibacterial propcrrics
demonstrable experimentally, their relevancr in rhe
natural context is not clearly understood.
A method of defence ag.iinst vital infections is
the production or :uterferor by cells sti-nulated by
live or killed viruses and certain other inducers.
Interferon has been shown ro be more i-nportanr
rhan specific antibodies in protection against and
recovery from certain acute viral infections. Tissues
and body secretion* contain other antiviral
substances.
Mi c ro h ia l an tagon izin g: The skin and
mucous surfaces have resident bacterial flora which
prevent enlcmlsailon by pathogens- Altera: ion of
normal resident flora may lead to invasion by
extraneous nil robes, causing serious diseases such
as staphylococcal or clostridial enterocolitis
following oral antibiotics. The importance of
normal bacterial flora ml native immunity i$
exemplified by the extreme susceptibility of germ
free animals to ail types of infections.
C e llu la r f a c t o r ! in in n a te im m u n ity ;
Natural defence against the Invasion of blo<id and
tissues by microorgani-.ms and other foreign
particles Is mediated to a large extent by phagocytic
eelb which ingest and destroy them. Phagocytic
cells, originally d^covercd by Mctchnikoff (1SS3),
were classified by him into microphages and
macrophages. Micro phages arc polymorpho­
nuclear leucocytes. Macrophages con-ist of
histiocytes which ate the wandering amebnid cell*
seen in issues, the fixed re^cubendotheliaJ cells
and the monocytes in the blood. A major fund ion
of the reticuloendothelial system is the removal of
foreign particles that enter the body. Phagocytic
cells reach the sites of inflammation in large
numbers, attracted by chemoiactic substances, and
ingest paniculate materials. Capsulared bacteria,
such an pneumococci, are not readily phagocytoscd
excepl In the presence of opsonin*. They are more
readily phagocytosed when trapped against a firm
surface such as the alveolar wall than when they
arc free in [issue fluids. Bacteria are phagocytosed
into a vacuole (phagosome), which fuses wirh the
lysOsomes round In the cell to form the
phagolysosome. The bacteria are subjected to the
action of the !y1:c enzymes in the phagolysosome
and are destroyed. Some bacteria, such as brucella
and lepra bacilli, n&ist intracellular digestion and
may actively mulriply inside the phagocytic cells.
PhagocytOMS in such instances may actually help
to disseminate infection to dirk run partis of the
hody.Th-e importance n1 phagocytosis in protection
against infection is evidenced by the entranced
susceptibility to infection seen either when the
phagocytic cell* are depleted, us m i^rflnulocytosi:-,
or when they are functionally deficient, as in
chronic granulomatous disease. A class of
lymphocytes called natural killer (NK) cells are
important in nonspecific defence against viral
infections and tumour*. They selectively hill vims
mfecred cells and tumour cells. NK cells are
activated by interferons.
I nllnnim ntinn: Tissue injury or Irritation,
iinitiated by rhe entry of pathogens or other irritants,
leads to i nfl animation, which i-i an important,
nonspecific defence mechanism. The arterioles at
the site constrict imti.dly and then dilate leading to
an increased h]ood flow. There is a slowing of blood
flow and margi nation of the leucocytes, which
CSCapt into rhe Li sSu Cs by i.Jiupedesis Jrid accumulate
in large numbers, attracted by the cberrotactic
substances released at the site of injury.
M icrooiganisms are phagpeyfosed and destroyed.
There is an outpouring of plasma, which helps to
C o p y rig h te d m aterial
dilute due toade produces present, A fibrin barrier is
hid, serving to wall off the site of Lnfection.
A rise of temperature following infection
is a natural defence mechanism. 31 not merely helps
to accelerate the physiological processes hut mav,
in some cases, actually destroy the infecting
pathogens. Therapeutic induction of fever was
employed for the destruction of Treponem a
pallidum in the tissues uf syphilitic patients before
jicnicillin became available. Fever stimulates the
production of interferon and aitk recovery from viral
infcctions.
In fee rim: or injury
leads to a sudden increase in plasma concentrations
of certain proteins, collectively called acute phase
proteins. iTcsc include C reactive protein (CJRP),
mannose binding protein, alp ha-1-acid
glycoprotein* scrum amyloid F component and many
others. CRP and some other acute phase proteins
activate the alternative pathway of complement.
They arc believed to enhance host resistance, prevent
tissue injury aitd promote repair of inflammatory
lesions.
The resistance chat an individual acquires during
life is known as acquired immunity as distinct from
inborn innate immunity. Acquired immunity is of
two types, active and passive. Active immunity is
the resistance developed by an individual as a result
of an antigenic stionulus. It is also known as ddapriVe
unmunrjy as it represents an adaptive response of
the host to a specific pathogen or other antigen.
This involves the active functioning of the host's
immune apparatus leading to the synthesis of
antibodies and the production of immunologically
active veils. Active immunity sets in only after a
ta tf nt period which is required for the
immunological machinery to he scr in motion.
During the development of active immunity, there
is often a negative phase during which the level of
measurable immunity may actually be lower than
it was before the antigenic stimulus. This is because
tire antigen combines with any pre-odsdng antibody
and lowers its- level in circulation. Once developed,
the active immunity is long-lasting. If in individual
who ban been actively immunised against an antigen
experiences the same antigen subsequently, the
immune response occurs more quickly and
abundantly than during the first encounter. This is
known ;lh secondary response. Resides the
development of humoral and cellular immunitv,
active immunity is associated with immunological
memory. 'I'his implies that the immune system ig
able to retain tor long periods the memory of a
prior antigenic aq u atic and ro produce a secondary
type of response when it encounters rhe same
antigen again, Active immunisation is more
effective and confers better protection than passive
immunisation.
The resistance that is uarLsmined passively to a
recipient in a 'readymade' form is known as passu1*:
immunity, Here the recipients immune system plays
no active role. There is no antigenic stimulus;
instead, preformed antibodies are adlirnmHteied.
Their is no latent period in passive immunity,
protection being effective immediately after passive
immunisation. There is no negative phase,. The
immunity is transient, usually lasting for days or
weeks, only till the passively transmitted antibodies
are metabolised and eliminated. No secondary type
response occurs in passive iintnunity. |n fact, passive
iin muni tv diminishes in effect with repetition.
When a foreign antibody it administered a second
time, li is eliminated more rapidly titan initially.
Following the first injection of an antibody such as
immune horse serum, the elimination is only hy
metabolic breakdown but during subsequent
injections ■if horse scrum, elimination is much
quicker is it combines with antibodies to horse
serum that would have been produced following
its initial injection. This factor of jjttifluric
elimination limits the usefulness of repeated passive
immunisation. Passive immunisation isles*effective
and provides an immunity inferior to that provided
by active immunisation. The main advantage of
C o p y rig h te d m aterial
76 * TexT&ook o1 MicratuDlogy *
Ti.1 i■■■:■ immunisation i- that H acts nmmeduteh1
and, therefore, can he employed, when 'instant'
immunity is desired (Table lO.lJ.
Active immunity may be natural or artificial.
Natural active immunity results from either a
clinical or nn inapparent infection byii microbe. A
per-'Ci who has recovered from an attack of measles
develops natural active immunity. I’hc hirgc minority
of adults in the developing countries possess natural
active immunity to poliomyelitis due to repeated
inappsrent infections wirh polioviruses during
childhood. Such immunity is usually long-la&ilng
hut the duration vanes with the type of pathogen.
The immunity is lifelong following many ^iral
diseases such as chicken pox or measles. In some
viral diseases, such as influenza nr common cold,
the immunity appears to be shortlived. Influenza
can recur in an individual after a few months or a
year hul fhi> is not sd much due to lack of the
immu nising capaci 1 y of the v ii us as to : ts abil iry to
undergo anti genie variation so that immunity
following the first infection is not cffectnT ags.inst
the seonnd jnfectk m caused by an antigC-1 i.' .111y novel
virus. In common cold, the apparent lack of
immunity is because rhe same clinical picture can
be caused by infect loti with a large number of
difiercn r viruses. '[“he 1mnrnnity follow 11 ig bactei ial
infection is generally less permanent than that
following viral infections. Some, such as typhoid
fever, induce durable protect 11ilL In syphili-, a special
type of immunity' known as 'premunitiem’ is seen.
1 lere, the immunity to reinfection lasts only as long
as the original infection rcm;iins active. Once the
disease is cured, the patient becomes susceptible to
the spirochete again. In chancroid, another venereal
disease, caused by Haemophilus ducreyi, there does
not appear to be any effect ivt 1rnmui 1icy an the pat iant
may develop lesions following reinfection even
while the original infection in active.
Artificial active immunity is the resistance
Induced by vao incs. Vaccines arc preparations of
1ivc or kil.led n1icrootgan isms or thd r products used
for immunisation.
Examples of vaccines are as follows:
1. H aclrrial vaccines
a. Live (BCG vaccine for tuberculosis)
b. Killed (Cholera vaccine)
c. Subunit \Fyphuid Vi antigen i
d. Bacterial products (Tetanus twadd)
2. Viral vaccines
a. Live (Oral polio vaccinc^Sabin)
k Killed (Injectable polio vaccinc-Salk)
c. Subunit {[ leparitis 13 vaccine)
f.ive vaccines initiate an infection without
causing any injury or disease. The immunity
following live vaccine administration therefore
parallels that following natural infection though it
may he of a lower order. The immunity lasts for
several years but booster doses may be necessary'.
Live vaocmes may be admi"listened orally' (as with
the Sabi 11 vaccine for poliomyelitis) or paientcrally
(as ivith the measles vac Line). Kilted vaccines arc
generally less immunogenic than live vaccines, and
prntecl ion lasts only lor a short period. They have,
therefore, to he administered repeatedly' generally
at least two doses being required for the production
of immunity. The first Is known as the prunuy
dose and the subsequent doses as booster doses.
Kilted vaccines may' he given orally hut this route
is generally not effective. Parenteral administration
provides humoral antibody response, which may
be improved by the addition of adjuvants1 (for
example, aluniinmm phosphatL-h
Natural passive immunity is the resistance
passivel \ transferred from mother 10 baby. In human
infants, maternal Antibodies arc transmitted
predominantly through the placenta, while in
animals such as pigs, transfer of amihodies occurs
mainly orally through the colostrum. The human
colostrum, which is also rich in IgA antibodies
resistant ro intestinal digestion, gives protection to
the neonate.The human fetus acquires some ability
to synthesise antibodies (IgM) from about the
tw en tieth Week o f life but its im m u n o logical
capacity' is still inadequate at birth. It is only by
about the age nl three m onths that th e Infant
C o p y rig h te d m aterial
 EJtoduced actively by host's immune system
Induced by infection or by immunogens
Durable effective protection
Immunity effective only after Lag period
Immunological memory present
Booster c-ffecr on subsequent dose
'Negative phase1 may occur
Not applicable in the immunodcficiem
acquires some measure of immunological
independence, Until then, maternal antibodies give
passive protection against infectious disUfCS to the
infant. Transport of antibodies across the placenta
is an active process and therefore the concentration
of antibody in the fetal blood may sometimes be
higher than that seen in the mother. Protection so
,l6tbrded will ordinarily be adequate againht all the
common infectious diseases in the locality. It is for
this reason that most pediatric infections are more
common after the age of three months than in
younger infants. Ely active immunisation of mothers
during pregnancy;, it is possible to improve the
quality of passive immunity in the infants.
Immunisation iff pregnant women with tetanus
toxoid in recommended for this purpose in
communiiicE in which neonatal tetanus is common.
Artificial pasjive immunity is the resistance
passively transferred to a recipient by the
administration o f antibodies. The agents used for
this purpose are hyperimmune sets iff anim;Ll or
hum an origin* convalescent sera ami ponied human
jrammi globulin. These are used lor prophylaxis
and therapy1. Equine hyperimmune sera such as
anti tetanus serum and ATS prepared from
hyperimmunised horses used to be extensively
employed. They gave Temporary protection but
carried the disadvantages of hypersennitivity and
immune elimination. Human hyperimmune
globulin {for example, tetanus im m une globulin,
TIG ) is free from those complications and also
gives more lasting protection. Antisera of animal
Received passively, No active host participation
Readymade antibody transferred
Transient, less effective
Immediate immunity
No mernuiy
Subsequent dose less effective
No negative phase
Applicable in immunudeticient
origin iirc now recommended only where human
preparations arc not available (anti gas gangrene
a ml and botulinum sera: antivenoms).
■Convalescent acre (sera [ff patients recovering
from infectious diseases) contain high levels of
Specific antibody R ni/clJ1 human gamma^pubuJin
(gammaglobulin from pooled sera of healthy adults)
contains antibodies against all common pathogens
prevalent in the region. Convalescent sera and
pooled human gammaglobulin were used for
passive immunisation against some virus infections
(like viral hepatitis A). I Inman gammaglobulin is
also used in the treatment of patients with some
immunodeficiencies. Gammaglobulin tends to
aggregate and when injected intravenously may
cause anaphylactic reaction due to complement
activation. They are therefore to be given only
intramuscularly. U has to be ensured that all
preparations from human sera are free from the
risk of infection wirh hepatitis U, hepatitis t\ HIV
and other infective agents.
Passive immunisation is indicated for immediate
and Temporary protection in a nonimiminc host
faced with the threat of an infection, when there is
insufficient time for active immunisation to take
effect. It is also indicated for the treatment ofsome
infections. Passive immunisal inn may also he
employed fbr rbe suppression of active immunity,
when the latter may be injurious. An example is
the UM of RJl immune globulin during delivery to
prevent immune response to the Rhesus factor in
RIl negative women with Rh positive bablCS-
C o p y rig h te d m aterial
7ft * Texiaook or Microbiology
Soni lCi:ii l a combination of uclive aftJ passive
methods of immunisation is employed. This is
know n ns c o m b i n e d j-mmun.'-tarron. Id e a lly
whenever passive immunisation is employed for
im mo. li.itc protect ion, enmhined immunisation is
to be preferred, as i o the protecti ■in o f a norti mmiin;
individual with a tetanus-prone wound. The
method is to inject TIG in one arm and the tint
dose of tetanus toxoid in the other This is fallowed
by the fill] course of phased tetanus toxitid injections.
TIG provides the protection necessary till the active
immunity is able to take effect.
A special type of immunisation is the injection
of immunoloyricallv competent lymphocytes. Tics
is known as adoprut jkrtrtumjry and does : kil have
general application. Instead of whole lymphocytes,
an extract of immunologically competent
lynlphucytes, known as the 'Transfer factor*, can be
used. Thi-- has been attempted in the Treatment of
certain types of diseases (for example leprnmarnus
leprosy).
M ea su rem en t of I m m u n it y
'Hie truly valid measurement of immuniry is to test
l]lL" resistance of an individu.il to a challenge by
the pathogen.Thi- m, however, not applicable since
the challenge itself alters the state of immunily. It
is, therefore, rot possible to measure accurately the
level of immunity in an individual. Estimates of
Immunity are generally made by statistical methods
using large numbers ot individuals.
A simple method of testing immunity is to relate
its Level to some convenient indicator, such as
demonstration of the specific antibody. This is not
always reliable as the immune response to a
pathogen consists of the formation of antibodies to
several antL^ens present in it, ait also to the
production of cellular immunity. The antibodies
maybe demonstrated by a vai iery of techniques such
as agglutination, precipitation, complement fixation,
hemagglutination inhibition. neutTulisati'in, I'll ,1SA
and others. In the absence of exact information as
to which antigen of the pathogen constitutes die
*
prorecrivie antigen, serological attempts Lo measure
immu ni:y are at best only approxi mations. In some
instances, as in diphtheria where pathugcncsti is
due to a well-defined antigen (the toxin), the level
of immunity can he assayed by in vitro or in vivo
(Schick test) methods. W here protection is
associated with ccll-mcdiatcd immunity, skin tests
for delayed hypersensitivity and in vitro tests for
CM ! afford an indication ot jitimunity.
L o <;a l I MMi NITK
The concept of local immunity, proposed by
Besredka (191^-74), has gained importance ir. the
treatment of infections which arc localised or where
iL is operative lit combating infection at the site of
primary entry of the pathogen. In poliomyelitis, for
instance, sysremit immunity provided by active
immunisation wi:h the lei ILed vaccine neutralises
the virus when ir enters the bloodstream, but it
docs not prevent multiplication of the virus at the
sire of entry (the gut mucosa) and its fecal sheddi ng.
Thi-- is achieved by the local iiitestirial immunity
acquired either as a result of natural infection or
immunisation wn:h the live oral vaccine. In
influenza, immunisation with the killed vaccine
elicits a humoral antibody response- But the
antibody title in tespiratnry secretion? is- often not
high enough to prevent infectii’iL Natural infection
or the live virus vaccine administered intranasally
provides local immunity. A special class of
immunoglobulins (IgA) forms the major
component of local immunity.
One type of IgA antibody called secretory IgA
is produced locally by plasma cells present on
mucosal surfaces or in secretory glands. There
appears CObe a selective transport of such antilxxiics
between the various, mucosal surfaces and secretory
glands. Thus, following intestinal exposure to an
antigen, the specific IgA anubody and the plasma
cells form ing such an an ribody can be demonstrated
in breast milk- This Indicates the existence of a
common mucosal or Memory immune system.
Besides providing local defence against
C o p y rig h te d m aterial
 * Immunity * 79

microorganisms, [he mucosal imitiunt system may
also be involved iri handling various antigens that
m ay came i:]to contact w ith muensai s-uriaces from
the external environment or through food.
H e r d [ m m u n fti
This refers to rhe overall level of immunity in a
community and is relevant in the control of epidemic
diseases. When a large propartnm of individuals in a
community (heed) ate immune to a pathogen, the
herd immunity m rhe pathogen k sim^factory. When
herd immunity is tow, epk lemur; air likely to occur
on the introduction of a suitable pathogen, due to the
presence of laig; numbers of susceptible individuals
in the community. Eradication of communicable
diseases depends on the development of a high level
of herd immunity rather than on the development of
a high level of immunity individuals.

Further Reading
JanewayCA and P Travers ISfM. Immiinobioia^y. London: Cumnc Riolngy.
Cabay C and I Kushntr IW*}. Aljuccphase pjemtins. \ EnglJ Mrd 34tM4H.
Kh riiocowsky D. SOW. Susceptibility t,- infeciiDn. Br M td _/^21:1 fWsI.
Rant IM. 1W4 E s s c n r i a f t f 11' edit. laindon: Rlfrln*ell Scientific.
Weir DM and_f Srrwarr 1997. f/njiNinaA^g-r. S* edn. Edinburgh: Churchill Livingitrine.

C o p y rig h te d m aterial
 Antigens
An antigen has been defined as any substance
which, when introduced paienteralh into the body,
st imutates the prod ucrion of an ant ibody with wl iich
it ft acts specifically and in an observable manner.
This traditional description of an antigen is on
longer comprehend1vc enough in the light of current
concepts about the immune response. Some antigens
may rot induce ant ibodi es but may sens iri-c spec ifk
lymphocytes leading to cell mediated immunity or
may cause immunological tolerance.
The word 'parenteral' (meaning, outside the
intestinal tract) is used in the definition because
orally administered antigens arc usually denatured
by digestive enzymes and their antigenicity
i lc s' njyct I. so that no an Lint dy format ion tabes place.
When given parenterally, antigens do not undergo
any such inactivation and can induce antibody
production. However, there axe exceptions and some
antigens can be immunogenic when given orally,
such as oral vaccines.
The word 'specifically’ in the definition is
important as specificity is the hallmark of all
immunological reactions. An antigen introduced
into the body reacts only with those particular
immunocytes (B or T lymphocytes) which cany
the specific marker for that antigen and which
produce an antibody or Cells Complementary to that
antigen only. The antibody so produced will react
only with that particular antiL^en and with no other,
though immunological cross reaction may occur
between closely related antigens.
The two attributes of antisjeniofy arc (1)
inducrlon of an immune response (immuno-
genicityj, and (2) specific reaction with antibodies
or sensitised cells (immunological reactivity). Based
on the ability to carry out these two functions,
antigens maybe classified into different types. A
com plete intigen is Able tu induce antibody
former ion and produce a spec: fie and observable
reaction with the antilwdy so produced. Haptens
are substances which are incapable of inducing
antibody formation by themselves but can react
specifically with amibodies. (The term hapten is
derived from the Greek hzptcin which means 'to
fasten’.) Haptens become immunogenic (capable of
inducing antibodies) on combining with a larger
molecule carrier. Haptens may be complex or
s11 nple; while complem haptens can prec ipirate wir!h
specific antibodies, srm/iJe haptens are
nonprecipitating. Fhey can inhibit precipitation, of
specific antibodies by the corresponding aTltili)Cn
or complex hapten. Complex and simple haptens
have been described as polyvalent and univalent,
respectively, since it is assumed chat precipitation
requires the antigen to have two or more antibody
combining sites.
[ in smallest unit of antigenicity in known as
the an^gcnic deternirnanf or epitope. The epitope
is that small area on the antigen, usually consisting
of four or five a mi noacid nr monosaccharide
residues, possessing a specific chemical structure,
elect]ical charge and stenc (spatial) configuration,
capable of sensiri-.ing an immunocyte and of
reacting with its complementary site on the specific
antibody or T-ceU receptor. Epitopes may be present
as a single linear segment of the primary sequence
(sequential or linear epitope) or formed by bringing
together on the surface residues from different sites
C o p y rig h te d m aterial
i)f tin: jicptitie d iu.3n during its folding into the
tcrtiarv structure (conformational epitope). 1 cells
recognise sequential epitopes, while H cells idenrity
the tertiary Cunfijipj ration of the conformational
epitopes- The combining area on the antibody
m olecule, corresponding to the epitope, is called
the }wmropCr Epitopes and paratopes determine the
specificity of immunological rcfurtinns, Antigens such
as bacteria or viruses cany many different types of
epitopes, presenting an antigenic mosaic.The presence
of tli? same or similar epitopes on different antigens
accounts for otw type of antigenic crow- reaction.
A number of properties have been identified which
make a substance antigenic but the exact basis of
antigenicity is still not cleat.
A n tig e n ic ity is related to m o le cular Eiae. V e ry
large molecules, such as heniocyanins (1VIW h,75
million), are highly antigenic and particles with
low m o le c u la r w e ig h t (less than 5 0 0 0 ) ate
non antigenic or teehty so. L o w molecular weight
substances maybe rendered antigenic by adsorbing
them on large inert particles such as bentonite or
kao lin . Some Low molecular weight substances
(such as pi cry [ chloride, formaldehyde and
penicillin] m ay be antigen in when applied on the
skin, pnohably bv com bining with, tissue proteins.
'Fhcv are haptens of Low im m unngenicity, effective
In same persons only and related to hypersensitivity.
Most naturally occurring
antigens are proteins and polysaccharides. Lipids
and nucleic acids are less antigenic. Their
antigenicity is enhanced by combination wirb
proteins. A certain degree of structural diversity is
required for antigenicity. That probably explains
why proteins which are composed of about 20
different a mi no acids ire better antigens than
polysaccharides which have only four or five
monosaccharide units. However, not all proteins
arc antigenic. A well-known exception is gelatin,
which is nonircirminogcnic because of its structural
i instability.
Only
substances which are metabolised and ate
susceptible to the action of tissue enzymes behave
as antigens. Antigens introduced into the body are
degraded by tlic host liLto fragments of appropriate
size containing the antigenic determinants.
Phagocytosis and m trice lliila j enzym es appear to
play an essential role in. breaking down antigens
into immunogenic fragments, Substances
unsusceptible to tissue ennmes such as polystyrene
Latex are not antigenic. Substances very rapidly
broken down by tissue enzymes are also not
antigenic. Synthetic polypeptides, composed of D-
aminoacids which are not metabolised in the body,
are TWt antigenic, while polypeptides consisting of
L-amtnoacids are antigenic.
Only antigens which arc “foreign1
to the in d iv id u a l (n o n s rll ) induce an ] mu m ile
response. T h e uni mat body con tains num erous
lantigenK w hich induce an im m une response when
introduced into another individual or species. An
individual does not normally m ount an immune
response ag ain st bis aw n normal cim sritu e n t
and gens-"Hus was first recognised hy E h rlic h who
proposed rhe concept of “horror autotoKieuS (which
means fear of self 'poisoning). Tolerance of self
antigens is conditioned by contact with them during
the development of the immune apparatus.
Breakdown o f t hiss homeostatic mechanism results
in autoim m unisation and autoim m une disease.
To general, the antigenicity of a substance is
related to The degree of its lore ignness. Antigens
from other individuals of the same species are less
antigenic than chose from other species. Antigens
from related species are less antigenic than chose
from distant species.
The basis of antigenic
specificity is stereochem ical, as was first
demons' rated by Obermayer and Pick and
confirmed by Landstciner. Using haptens such as
atoxvl couplel) with protein, :L was shown that
antigenic specificity is determined by single
chemical groupings and even by a single acid
C o p y rig h te d m aterial
radical. The importance ot the ]>l>slClc?rt of the
antigenic determinant group in the uritj^cn
molecule was evidenced by the differences in
specificity in compounds with the group attached
at the ortho, mefn fir para positionH.Thu influence
of spatial configuration of the determinant group
was shown by differences In antigenic specificity
of the dextro, leva and mesa isomers o f substances
such as tartaric acid.
Am iodine specificity is not absolute. Cross
reactions can occur between antigens which hear
stereochemical similarities- In some instances,
apparent cross reactions may actually be due to the
sharing of identical antigenic determinants by
different antigens.
The Specificity o f natural tisiue antigens of
animals may be considered under diflvrent categories
as species, iso-, auro- and organ specificities,
Tissues of all individuals
in a secies, contain species-specific antigens. There
exists, some degree of cross "reaction between
antigens from related species. This immunological
relationship parallels phylogenetic relationships. It
has been used in tracing evolution ary relationships
between specif:?. |t also has forensic applications
in the identification of the species of blood and of
se m in a l stains. P h y lo g e n e tic re latio n sh ip s are
reflected in the extent of cross reaction between
antigens from different species that cause
hypersensitivity. An individual sensitised to horse
semm wilt react with serum from other equities
but may not do so with bonne serum.
Isoantigens arc antigens found
in some bur not all members of a species, A species
may be grouped depending oti the presence o f
different isoantigens in its members. The best
examples ol isoantigens are the human ent hrucyte
antigens based cm which individuals can be
classified into different blood groups. 'These are
genetically deter mined. They are oi clinical
importance in blood transfusion and in
tsoimmunisation during pregnancy. They were of
help in determining disputed paternity canes, but
have been supplanted by the more discrimiuatoiy
DNA fingerprinting tests, Blood groups find
application in anthropology.
Histocompatibility antigens arc those cellular
determinants specific to each individual of a species.
They arc recognised by genetically ditferent
individuals of the same species when attempts are
made to transfer or transplant cellular material from
one individual to another [described in Chapter
15).
Autologous or self antigens
ure ordinarily nonandgeflic bur rbere are exceptions.
Sequestrated antigens that are not normally found
free in circulation or tissue fluids (such ns eye lens
protein normally confined within Its capsule) arc
not recognised as self antigens. Similarly, antigens
that are absent during embryonic life and develop
later {such as sperm) arc also not recognised as self
antigens.
Some organs, such as the
brain, kidney and lens protein of different species,
share the same antigen. Such antigens, characteristic
■of an organ or tissue and found in different species,
arc called o rg an specific antigens. The
neuroparalytic complications following antirabiv
vaccination using sheep brain vaccines arc a
cumcijuencC of brain specific antigens shared by
sheep and hum an heings. The sheep brain antigens
induce immunological response in the vaccinees,
damaging their nervous tissue.
'The same or closely related antigens may sometimes
occur in different biological species, classes and
kingdoms. These are known as heterogeneric or
heterophil* antigens, best exemplified bv the
Fornsman antigen which is a lipid carbohydrate
complex widely distributed in many animals, birds,
plants and bacteria- It is absent in rabbits, so anti-
Forssman antibody can be prepared in these
animals. Other hetemplhde antigens are responsible
for some diagnostic serological reactions in which
antigens unrelated to etiological agents are employed
(hetemp bile rc action). The Weil—Felix reaction in
C o p y rig h te d m aterial
 i A n ttg e n a * fl3

typhus fever, the Pau] Runnel test in infectious
mononocleo*i■) and the cold jgglutinin test in
primary atypical pneumonia are examples.
liLDLOGICAL CLASHES OF ANTIGENS
Depending on their ability to induce antihody
formation, antigens are classified asT cell dependent
(T D ) and T cell independent (T I) antigens.
Antibody production is the property of B
lymphocytes. For the full expression of this function,
however, the cooperation of T lymphocytes is
necessary. Some antigens can directly stimulate
antibody production by R cells, without the apparent
parti( ipadon of T cells. Such antigens are called TI
antigens. Others that require T cell participation to
generate an immune response are called TD antigens.
Several im portant differences exist between
TI and T D antigens. TT antigens arc structurally
sim ple, beulg composed o f a lim ited num ber o f
re p e a tin g e p i'o p c s , as in th e case o f fbc
pneumococcal capsular polysaccharide, bacterial
1ipopolyaacehnudes and the flagellar protein
flagcllin. Their Immune response is critically dose
dependent. Ttto little i>non imimmogenk, while rt10
much results in immunologi^l tolerance rather
than immunity. Their antibody response is usually
limited to IgM and IgG3. They do not show
immunological memory TI antigens do not appear
to require preliminary processing by macrophages.
They arc metabolised very slowly and remain in
the body for long periods.
T D am Lien's, on the other hand, are structurally
more complex, such as erythrocytes, scrum proteins
and a variety' of protein-hapten complexes. They
are immunogenic over a wide dose range and do
not cause tolerance readily. T h e y induce the fiill
gamut of immunoglobulin isotypes— IgM. JgG, IgA
and IgE, They' show immunological memory,
require preliminary processing, and are rapidly
metabolised in the body.

I' 11 rther t t n d i n ^
DM .■■■-1 I Stewart 1997. Jmmunvivgf. 9th rdn Edinburgh; Churchill Livingstone.

C o p y rig h te d m aterial
immunoglobulins, but all immunoglobulins may not
be antibodies.
Immunoglobulins constitute 20-25 per cent of
the rota! serum proteins. Based on physicochemical
and antigenic differences, five classes fit
immunoglobulins haw been recognised— IgG, IgA,
IgM, TgD and TgE. (Both Ig and y art accepted
abbreviations for immunoglobulins).
Studies involving the cleavage of rite
immunoglobulin molecule, pointertd by Porter,
Edebnan, N iso n o fi and their colleagues, have led
to a detailed picture of its structure. Rabbit IgG
antibody to egg albumin, digested by papain in the
presence of cysteine, was split into two fractions—
an insoluble fraction which crystallised in the cold
[called Fc for cnutailix.iblt:}, and a snluhlc frag me of
which, while unable to precipitate with egg
albumin, could still bind with it. Tbis fragment lh
called the Fab fand^en binding) fragment- Each
molecule of Immunoglobulin it split be papain into
three parts, one Fc and two Fs6 pieces, having a
sedimentation coefficient of 3.5 S. When treated
T-vitJi pepsin, a 5 S fragment is obtained, which i*
composed essentially of two Fab fragments held
together in position. It in bivalent and precipitates
with the antigen. This fragment is called Ffabr)2-
Thc F c portion is digested by jjcpsin into smaller
fragments (Fig. 12.2).
immunoglobulins ire glycoproteins, each
molecule consisting of two pairs of polypeptide
chains of different sizes. The smaller chains art
called 'light' fId chains and the larger one? 'heavy'
ill) chains. The L chain has a molecular weight
ol approximildly 25„000 and the H chain ot 50,000.
The L chair in attached tn the H chain by a
dinulphidL: bond. The two H chains arc joined
together by 1-5 b-S bonds, depeiiding on the class
of immunoglobulins (Fig. 12.3)..
C o p y rig h te d m aterial
 The H chains arc structurally Lind antigenieally
distinct for each das?, and arc designated by the
Greet letter corresponding to the immunoglobulin
dass as shown below:

ikg y (gamma)
IgA a (alpha)
IgM fl (mu)
IgD & (delta)
M ___________ C (epsilon)
T h e L ch ain * are s im ila r in ail classes of
immunoglobulins. They occur in two varieties,
kappa ( t ) and lambda i>d. A molecule of
immunoglobulin rmay have cither kappa or lamhda
chains, bur never both together. Kappa and lambda
are named after Korngold and Lapsiri who originally
described them. T h e kappn and lambda chains occur
in a ratio of about 2:1 in human sera.
T h e antigen com bining site o f die molecule is
jiits am inorerm inus. It is composed o f both L and
H chains. Studies on the prim ary structure of L
and H ch a in i show that they consist o f two portions ' 1■ l .... • I./ i
each. O f the 2 1 4 am innucid residues that make up
th e 1 1 c h a in , ab o u t 1 0 7 th at c o n s titu te the
<v:" [ ■; ::j“ : t i r f-f; / : ’ T VV
carbofiytcrm inal half, occur o n ly in a constant lh j CHislanl raflk i {CV srMli sitch J :avy evjeIi
sequence. T h is part o f the chain is therefore called Ini' v * .r ,,. iita c ; . " : i , i j - ,u t . c :■?
the 'constant region'. O n ly two sequence patterns and t: ..vs! civ kvJ Civ \ ihr< - ■\yi rs* tie.
3 re seen in the constant region - those determining I he am im; acid sequences of the variable rtjpnns
the kappa and lambda specificities. On the other of the L and H chains ate not unitbrmly variable
hand, the aiTurtCUcid sequence in ih c ai mi ml cm mini ■along tlieif le ngfli, but consi st ■1 1re Iat ivcly invariable
h a lf o f the chain is highly variable, the variability and some highly variable zones. Tfet highly variable
determ ining rhe im m unological specificity o f the iwnes numbering three in the L and four in the H
■antihody molecule. It is therefore called the Variable chains ate known .is hypervuri able regions (or hot
region, llic H chair also has 'constant' and Variable' -piitsl and arc involved with the formation oi'ihc
regions, Wltile in dw L chain the two regions are o f antigen binding sites.Tltt: sites on the hvpervariable
equal length, in the H chains Ilie variable region regions Ihat make actual contact with the epitope
constitutes approximately only a fifth of the chain and arc L-slled complcmcntBiity determining regions'
is located at its aminotcnnlmia. The infinite range o f or CDKs.
the anrilxjdv sfiecificilv of immunoglobulins dejiendt; rile Fc fragment is Composed ot the
on the variability ot the luninoacid sequences at the carbnxvteiminal portion or the II chain-.. It doe-,
Variable regions' of the H and L chain-. which ftum not possess an tlgco combining activity Inn
the antigen combining sites. determines the biological properties of the

C o p y rig h te d m aterial
Litimuiinglubulm molecule such as complement
fixation, placental tin ns for, skin fixation and
catabolic rite. The portion of the H chain present
in the Ffrh fragment Lg called the Fd piece. The H
chain carries a carbohydrate moiety which is distinct
for each class of immunoglobulins.
Each immunoglobulin peptide chain has
internal disulphide links in addition to interchain
disulphide bonds which bridge the H and [_ chains.
These interchain disulphide bonds form Loops in
the peptide chain, and each of the hops is compactly
folded to form a globular domain,, each domain
having a separate function. The variable region
domains, VL and VH, arc responsible for the
formation of a specific antigen binding site. The
C H J region in JgG binds C lq in the classical
complement sequence, and the C H J domain
mediates adherence to the monocyte surface. The
areas of the H chain in the C region between the
flrnt and second C region domains (C H : and CHJ)
is the hinge region. If is more flexible and is mote
exposed ro enzymes and chemicals. Papain acts here
to produce one Fe and two Fab fragments (Fig.
12.3),
Human sera contain IgG, IgA, IgIVI, Igl) and IgU
in the descending order o f concentration. "fable
J2.1 shows their charactcristics.
This is the major serum immunoglobulin,
constituting about SO per cent of the total. It has a
molecular weight of 150,000 (7$), IgG may
occasionally exist in a poly me fined form. It is
distributed approximately equally between the
intravascular and extravascular compartments. It
contains lens Curb oil yd rate thin other
immunoglobulins. It has a half-life of
approximately 23 days. The catabolism o f IgG is
unique in that it varies with its scrum concentration.
When its Level is raised, as in chronic malaria, kala
AZar Or myeloma, the IgG synthesised against a
particular antigen will he caiabolised rapidly and
may result in the particular antibody deficiency.
Conversely, in hypogammaglobulinemia, the IgG
given tor treatment will be cafaboliscd only slowly.
The normal serum concentration o f IgG is about
8-1 fi mg per ml
IgG is the only maternal immunoglobulin that

Sedimentation coefficient (5) 7 7 19 7 8

Molecular weight 150,000 160,000 900,000 180,000 190,000
Serum concentration (mg/ml) l2 2 1.2 0.03 0-00004
Half life Wavs) 23 t, S 3-8 1 -5
Daily production [mgfkg) 34 24 3.3 0.4 0.0023
Ini KLviL'-vLihir distributum {per cent) 45 42 flO 75 50
Carbohydrate {per cent) 3 S 12 13 13
Complement fixarian
Classical - Ht - -
Alternative ~ 4- - - -

Placental transport 4- - - - -
Ihesent in milk + + - - -
Selective secretion by
seromucus glands - +■ - - -
Hear stability (56 °C) 4- ■* 4- «■
* IgA miy otmr m 7S, mid ITS farms.

C o p y rig h te d m aterial
is normally Transported i t ToSs the placenta and glycopcpride termed rhe J chain (J for joining).
provide* natural passive immunity in the newborn, This is called the secretory IgA (SlgA). Dimeric
It is not synthesised by the Ictus in any significant SIgA is synthesised by plasma ceils situated near
amount. IgG birds to microotganihiHind enhances the mucosal ot glandular epithelium. The j chain
their phagocytosis. Eictnictllular killing of target is also produced in the same cells. J chains are
cells coated with IgG antibody is mediated through present also LI] Other polymeric immunoglobulins
recognition of the surface Fc fragment by K cells such as IgM (Fig. 12.4}.
bearing the appropriate receptors. IutHSCton ol IgG S lg A Contains another glycint rich polypeptide
complexes wirli platelet Fc receptors probably leads called the secretory component - u secretory piece.
to aggregation and vasoactive amine rL-lcase. IgG This. is not produced by Lymphoid cells but bv
alone, among human iniimiroglnhiilina, can fix itself mucosal or glandular epithelial evil- Dimeric Ig A
to guinea pig skin, but the significance ot tbi* binds tn a receptor on die surface of the epithelial
property is not known, IgG participates in most Cells a] id ]>■ endocytosed and ■run -po rTe-■across the
immunological reactions such as complement cells to the luminal surface, I >■ tin this process, a
fixation, precipitation, Utd neutriLlisarinn of matins part of the receptor remains attached to the Ig A
and viruses. Jt may be considered a general purpose dimer. This part is known .l-- the secretory
antibody, protective ugamst those in factious agenfs component. The secretory piece is believed to
which arc active in the blood and tissues. Passively protect I g A from, dcnituration by bacterial
administered IgG suppresses rhe homologous proteases in sites such sis the intestinal mucosa
antibody synthesis hy a feedback process. This which have a rich and varied bacterial flora, S lg A
property is utilised in the isoimmunisatioft of women is a much larger molecule than serum IgA (11S;
hy the administration of anti-Khfl’1) IgG during M W about 400,000).
delivery, With most antigens, IgG is a late antibody SlgA is selectively concentrated in secretion*
and makes its ap] learunct: after the initial immune
response which is IgM :in nature,
Four subclasses of IgG have been recognised
(IgGI, lgG 2, lgG 3, IgG 4), c:ich possessing ,1
distinct type of gamma chain, identifiable with
specific antisera. The four IgG subclasses are
distributed in human serum in the approximate
proportions of 65 per cent, 23 per cent, 8 per cent
and 4 per cent, respectively,
Ig A i* the second most abundant class,
constituting about 1 0 -1 3 per cent of serum
immunoglobulins. The normal serum level is O.b-
4.3 mg per ml. It has a half lite of 6-B days. Tt is
the major immunoglobulin in the colostrum, saliva
and tears.
IgA occurs in two forms. Scrum IgA is
principally a monomeric 7S molecule (M W about
160,000}, IgA found on mucosal surfaces and it]
secretions is a dimer formed by two monomer units
joined Together at their carboxylerminals by a

C o p y rig h te d m aterial
anti on mucus surfaces forming an 'antibody pastr'
and is be Iiewd ro play an important role in local
immunity against respiratory and intestinal
pathogens. Secretory IgA is nelatiwlv resistant to
the digestive enstymes and reducing agents. IgA
antibodies may function by in bin iting the adherence
ot microorganisms to the Surface of mucosal cells
by covering the organisms and thereby' presenting
their ctitrv into body tissues. IgA does not tiX
complement hut can activate the alternative
complement pathway. It promotes phflgocvrosis and
intracellular killing ot microorganisms.
Two IgA subclasses have been described, IgA.
and IgAj. IgAj lacks interchain disulphide bonds
between tile heavy aod light chains. Though IgAj
is- j minor component of serum IgA, It is the
dominant form in the seerttiOrtS-
[gM constitutes 5-B per cent of sienim
immunoglobulins, with a normal level of 0-5-2 mg
per ml. It has a half-life of about five days. It is a
heavy molecule (19S; mol, m 900,000 to 1,000,000,
be licc called "the millionaire molecule'). IgM
molecules are polymers of five tbur-pcptidc subun iCS,
each bearing an extra CH domain (Fig. 12.5). As
with IgA, polymerisation of the subunits Spends
upon the presence of the J chain. Though the
theoretical valency is ten, this is observed only with
small haptens. Witli larger antigens, the effective
valency falls to five, pmhably due to stork hindrance.
Most of IgM (80 per ccnr) is intravascular in
distribution. Phylogenetically, ]gM is the oldest
immunoglobulin class. It Is also the earliest
immLinoghihuLin to be synthesised by [he fetus,
beginning by about 20 weeks of age. As it is nut
transported across the placenta, the presence of IgM
in the fetus or newborn indicates intrauterine
infection and its detection is useful in the diagnosis
of congenital infections such as syphilis, rubella,
HIV infection and toxoplasmosis. IgM antibodies
are relatively short lived, disappearing earlier than
IgG. Hence, their demonstration in serum indicates
recent infection. Treatment u£ scrum will] (X12M
2 - mcrcapfoethanol selectively destroy* IgM
without affecting IgG antibodies. This is a simple
method for the dltTcrcntinJ estimation of IgG and
IgM antibodies,
The isohemigglurinitis (anil-A, am i-13) and
many other natural antibodies to microorganisms
art usually IgM, as also antibodies co typhoid 'O’
antigen (endotoxin) and reagin antibodies in
syp hi! is.
The unique structural features of IgM appear
particularly suited to the biological role of
providing protection against microorganisms and
other large antigens that haw repeating antigenic
determinants on their surface, A single molecule
of IgM can bring ahout immune hemolysis,
whereas 1000 IgG molecules are required for the
same effect. IgM is also 500-1000 times more
effective than IgG in opsonisation, 100 times more
effective in bactericidal action acid about 20 Times
in bacterial agglutination. In the neutralisation uf
nrains and viruses, however, it is less active than
IgG. Being largely confined to the intravascular
space, IgM is believed to be responsible for
protection against blood invasion by
microorganisms. IgM deficiency is often associaled
with se puce mils.
Monomeric IgM is the major an ribody receptor
on the surface of B lymphocytes for Antigen
recognition.
C o p y rig h te d m aterial
90 * Tertbook of Microti iclog-y ►
IgD resembles IgG structurally. Lt is present
in a concentration of about 3 mg pet 100 ml of
serum and i$ mostly intravascular. It has a half-life
of about three days. IgD and IgM occur tin the
surface of uustimulared B lymphocytes and serve
as recognition receptors for antigens. Combination
oi cell membrane bound IgD or IgM Wi IH the
corresponding antigen leads to specific stimulation
of the R cell— enher aclivaiion and cloning It]
produce antibody, or supprest-i on.
IgFh This iimnnnoglobiiltn was discovered in 1966
by Ishizaka during tire investigation of atopic reagin
annhoclies. It is an SS molecule (M W about
190,000), with a half life of about two days. It
resembles IgG structurally. Jr exhibits unique
properties such as hear lability (inactivated at 56 °C
in one hour) and affinity lor the surface o f : issue
cells (particularly mast cells) of the same species
(homocytotropism). It mediates the Prausnitz-
Kustncr reaction. It is susceptible to
nrercaploethanot. It does ntit pass the placental
bar iicr or lot complement. It is mostly extntvascukr
in distribution. Normal scrum contains only traces
{a tew nanograms per ml) bur greatly elevated levels
are sect-, in atopic (type 1 allergic) conditions such
as asthma, hay fever and eczema. Children living
m insanitary conditions, with a high lu.nl of
intestinal parasites^ have hi^h serum levels of lg£.
JgE is chiefly produced in the linings o f the
respiratory and intestinal tracts. JgE deficiency bus-
been avsuCi.ited with IgA deficiency in individuals
WIlh nUpinred immunity who present undue
suscepcibilirv to infection.
IgE is responsible for the anaphylactic type of
hypersensitivity. The physiological role of IgE
appeara to be protection against pathogens by mast
cell dcgranulation and release of inflammatory'
mediators. Ec is also believed to have a spcci.it role
in defence agonist helminthic infections.
In general, IgG protects the body fluids, IgA the
body surfaces and IgM the bloodstream, while IgE
mediates magi ilie hypenens iti '.in'. JgD is a iec<ignition
molecule on the surface of B lymphocytes.
A bn o rm al I m m u n o g l o b u l in s
Apart from antibodies, other structurally similar
proteins ate seen : n scrum in many pathological
processes, and sometimes even in healthy persons.
I he earliest description of an abnormal
immunoglobulin was the discovery by Bence Jones
(1847) of the protein that bears hii name. Bence
Jones protein is found typically in multiple
myeloma. It can be identified in urine by its
characteristic property of coagulation when heated
to 50 °C but redissolving at 7Q ?C- Bcncc Jones
proteins arc the light chains of immunoglohulins
and so may occur as the kappa or lambda forms.
But ill any one patient, the chain is either kappa or
i.ui)hJ\t only, and never both, being uniform in all
other respects also, Tbi^ is because myeloma is a
plasma cell dyscrasia in which there is unchecked
proliferation of one clone of plasma cells, resulting
in the eveessivt production of the particular
immunoglobulin synthesised by the done. Such
iimmunoglobulins are, therefore, called monoclonal.
Multiple myeloma may affect plasma cells
synthesising IgG , IgA, IgD or IgE. Similar
involvement of IgM producing cells is known as
WaldenSWum > ittaLTO^fobuirrieni i.j. In Th is
condition, there occurs races*1vc production of the
respective myeloma proteins (M proteins} and of
their light chain* (Bence Jones proteins}. A different
disorder ip found in 'heavy chain disease', which is
a lymphoid neoplasia characterised by the
overproduction of the Fc parts of the immuno­
globulin heavy chains.
Cryogtohulincmia is a condition in which there
is the formation of a gel or a precipitate on cooling
the serum, winch redissolvts on warming. It may
not always be associated with disease but is often
found in myelomas, macroglobulinemiis and
autoimmune conditions such as systemic lupus
erythematosus. Most cryoglobulins consist of either
IgG, IgM or their muted pxeL iiutates-
Bccausc of the monoclonal nature ui’ Bence
Jones and other M proteins, they have been
C o p y rig h te d m aterial
 * AfinOOdiOS— l^mmur'iG^Dbirifl- 1 91

valuable models For the understanding of specificities. Antigenic specificities which

immunoglobulin structure and function.
I m m LNOGLOBUI t\ Sl>tiC lFtC m ES
The immunoglobulin specificity of the greatest
biological importance is idiotypic specificity
pcrtui ni ng to the nature of the antigen b itiding *itcs
(paratopes). The specific antigenic determinants on
the paratope arc called idiottrpes. The sum total of
idiotopcs on an lg molecule constitutes its idiotype.
By immunisation with Fab fragments*
anti idiotypic antibodies can be produced, These
resemble the epitopes of the original antigen. Used
as a vaccine, these show protean on against the
original antigen (pathogen or tumour) in
experimental animals. Sequential antiidiotypic
antibody formation is the basis of Jerne’s network
hypothesis of immune regulation.
Immunoglobuiius exhibit other genetically
determined speciticitins based on their am'igenic
structure. The antigenic specificities which
distinguish between the different classes and
sLihdasHes ol immunoglobulins present in all normal
individuals of a gjiven species are termed isotypic
distinguish immunoglobulins of the same class,
between different groups nf individuals in the same
species, are called allotypic specificities*
Immunoglobulin allotypes have been studied in
detail m the rabbit and guinea pig by using type-
specific immune Sera. Such deliberate immunisation
is not possible in human beings, but anualiorype-
spccitic antibodies may develop following blood
tranfusion or passage of maternal IgG into the fetus.
Antullotypc antibodies are atso found in sera
containing "rheumatoid arthritis factor.
Two allotypic systems are known in humans -
the Gm system (for gamma marker) and the InV
system (abbreviation of patients name). 'Fhe Gm
i^ associated with the Fc portion of the IgG heavy
chain* More than 25 Gm types have been identified
so far. The InV system is associated with the kappa
light chain and so has been renamed Km. Three
Km allotypes have been identified.
Genetic markers associated with IgA are called
Am\ To date, in the human system no allotypic
markers have been found for lambda light chains
or |A, 6 or e heavy chains.

Fu rth er Rending
(?■*.■. In: .r: JW. 1991. lriiinLi::Mi:lii|i'.Lli:: structure and i.nk r:i . V'1, edn. In Siuic and Cii'n,*aJ Immunology.
Rij-jrt IM and O Delves cd. V¥f2. EfHyclopedia o f Immunology: London: Academic Press.

C o p y rig h te d m aterial
 Antigen— Antibody Reactions
Antigens anti antibodies, by definition, combine
with ciich other specifically and in an observable
m anner. The reactions between IrtligtTLy and
antibodies serve several purposes. In the body; they
form the basis of antibody mediated immunity in
infectious diseases, or of tissue injury in some types,
of hypersensitivity and autoimmune diseases. In
die laboratory, they help in rite diagnosis of infections,
in epidemiological surveys, in the identification of
infectious agents and of noninfectLous antigens such
as enzymes. Ill general, these- reactions can be used
for the detection and qu^dtitation of either untigens
or antibodies. Antigen antibody reactions in vitro
are LrtOWrt as sefttkigical iH f M lt S
The reactions between antigens and antibodies
occur in three stages.The primary stage is rhe initial
interaction between the two, without any visible
effects. This reaction is rapid, occurs ever at low
temperatures .md obeys the general laws of physical
chemistry and thermodynamics. The reaction is
reversible, the combination between antigen and
antibody molecules being effected by the weaker
intermolecukf forces such as Van der Waal's forces,
ionic bonds and hvdrogm bonding, rather Than by
the firmer cowdent bondi]ig. The primary reaction
can be -del ecred bvendo ian ng free and Ix ji md a ntigen
or antibody separately in rhe reaction mixture by a
number oJ physical and chemi-tal m eth o d s,
including the use ol markets such as radioactive
isotopes, fluorescent dyes ur ferritin.
In most instances, but riot all, the primary stage
is. followed bv die secondary stage, leading to
demonstrable events such as precipitation,
agglutination, lysis of cells, killing of live antigens.
neutralisation o f r<wins and other biologically active
antigens, fixation of complement, immobilisation
of m o tile organ ism s and e n h a n c e m e n t ot
phagocytosis. W h en such reactions were discovered
Oiie bv t>rte, il was believed that a different type of
antibody was responsible for each type o f reaction
and the antibodies came to be designated by the
reactions they were thought to produce. Thus, the
an tibo d y c a u sin g a g g lu tin a tio n was called
that causing precipitation p recif> iiin t and
so on, .md the correspond] mg antigen, a g g h i t in o g c o ,
prcdpilrntgcn, and so on. By the 1920s, this view
was. replaced, by Z in s s e rs Unitarian hypothesis
which held that an antigen gave rise to only one
antibody, which was capable o f producing all the
different reactions depending on the nature o f the
antigen and the conditions o f the reaction. Roth
tbeyL- extreme views are fallacious. W h ile it is true
that a single antibody can cause precipitation ,
agglutination and m ost o f the other serological
reactions, it is also true that an antigen can stimulate
th e p ro d u ctio n of d iffe re n t classes of
im munoglobulins which differ in th eir reaction
capacities as well as in other properties [Table 1 3 .1J.
Srune antigen—antibody reactions occurring in vivo
initiate chain reactions that lead to neutralisation or
desrruct inn Lo f injurious in iLigens, i it to tissue damage.
ITese an; Tertiary' reactions and include humonaE
imin 111 uty against infectious diseaseas ad well at clinical
alleigy Li! id other immunological diseases.
UJJLIL ■3-jfe'TU3* il' ,', r
A n tig en -an tib od y reactions have the follow ing
general characteristics:
C o p y rig h te d m aterial
 Precipitation Strung
Agglutination Weak
Complement fixation Strung
1. The reaction is specific, an antigen combining
only with its homologous antibody and vice
versa, 'Phe specificity, however, is not absolute
and 'cross-reactions1 may occur due to antigenic
similarity or (elatedness,
2 . En ti re molecules react and not fragments When
an antigenic determinant present io a large
molecule or on a ‘carrier' partide reacts with
its antibodv, whole molecules or particles arc
agglutinated.
3. ’ntcre is no denafuration of the antigen or the
antibody during the reaction.
4. The combination occurs at the surface, lliereforc.
It is the Surface antigens, that are
immunolngically relevant. Antibodies to the
surface antigens of infectious agents arc
generally protective.
5 . The combination is firm but reversible. The
firm ness o f the un ion is influenced by the aifin itv
and avidity of the reaction, Affin ity refers to the
intensity of attraction between the antigen and
antibody molecules. It is a function of the
closeness of fiE between an epitope and [he
antigen combining region of its antibody.
Avidity is the strength of the btnul after the
formation of the antigen-antibody completes.
It rc fleets tlic overall combining property' of the
various antibody molecules in an antiserum,
possessing different affinity constants with the
multiple epitopes of the antigen-
6. Both antigens and antibodies participate in the
formation of agglutinates or precipitates.
7. Antigens and ant ibodscs can combine i n varyi ng
proportions, unlike chemicals with fixed
valencies, Roth antigens and antibodies are
multivalent. Antibodies arc generally bivalent,
Weak Variable
Strong Moderate
Weak Negative
though IgM molecules may have five or ten
combining sites. Antigens may have valencies
up to hundreds.
Many methods sre available for the measurement
of antigens and antibodies participating in the
primary, secondary or tertiary reactions.
Measurement maybe in terms of mass (for example,
mg nitrogen) or more commonly as unite of dire.
The antibody ritre of a scrum is the highest dilution
of the serum which shows an observable reaction
with the antigen in the particular tes-f. The title of
a serum is influenced by the nature and quantity' ot
the antigen and the type and conditions of the test.
Antigens may also be titrated agal nst sera.
Two important parameters of serological tests
are Sensitivity and specifttiTy. Sensitivity refers to
the ahilily of the test to detect ever very minute
quantities of antigen or antihody. When a test is
highly sensitive, fake negative results will be absent
or minimal. Specificity refers to the ability of the
test to detect reactions between homologous
.antigens and antibodies cjiiIv, and with no Other. In
a highly specific rest, folsc positive reactions are
absent or minimal, lit general, sensitivity and
specificity of a test arc in inverse proportion.
Originally, reagents for serological tests were
prepared by individual laboratories, leading to batch
variation, and lack of reproducibility and
compatibility. The commercial availability of
ready-made standardised test kits has simplified test
procedures, improved quality and greatly enlarged
their scope and use.
C o p y rig h te d m aterial

P R B C IP ' TA TIQ N & B A C TIO N
When a soluble antigen combines with its antibody
in the presence of electrolytes (NaCE) at a suitable
temperature and pH, the antigen-antibody complex
forms an insoluble precipitate. When, instead of
sedimenting, the preap itale remains suspended as
floccules, the reaction is known jr fJoccuktion.
Precipitation can take place in liquid media or in
gels such as agar, agarose or pulyaajiimide.
The amount of precipitate formed is greatly
influenced by the relative proportions o f antigens
and antibodies. If increasing quantities of antigens
are added to the same amount o f antiserum in
different tubes, precipitation will be found to occur
nuret rapidly and abundantly in one of the middle
tubes in which the antigen and uritiliudy are present
in optima] or equivalent proportions. In the
preceding tubes in winch the antibody it lii excess,
and in the later tubes in which the antigen is in
cvccrs the precipitation will be wr? k i>r even ah$en r.
For a given antigen^antibodv system, the optimal
or equivalent ratio will he constant, irrespective o f
the quantity of the reactants. If rhe amounts of
precipitate in the different tubes art plotted on a
graph, the resulting curve will have three phases:
an ascending part [prozone or zone of antibody
excess), a peak [zone of equivalence'; and a
descending part {posizoneor zone -it antigen excess)
(Fig, 13.1). This is called the zone phenomenon.
Zoning occurs in agglutination and some other
serological reactions. The pnnone is of importance
in clinical serology, u sera rich m antibody may
sometimes give a false negative predpication or
agglutination result, unless several dilutions urc
tested.
M e c h a n is m of P r e c ip it a t io n
Marrack (I^A ) proposed the lattice hypothesis to
rxplui n the mechanism of precip irati. 111 According
to this concept, which i* supported tu considerable
experimental evidence and is now widely accepted,
m u ltiv a le n t a n tig e n s coin b in t w ith b iv a le n t
C o p y rig h te d m aterial
 Eft'Jl-
antibodies in varying proportions, depending on the
antigen^antibody ratio in the reacting mixture.
Precipitation results when a large lartiee is formed
consisting nf a] tern a ting antigen and antibody
molecules. This is possible only in the zone of
equivalence. In the zones of antigen or antibody
excess, tile lattice does not enlarge, as the valencies
of the antibody and the antigen, respeelively, are
tulEy satisfied (Tig. 1.1.2) The lattice hypothesis
holds good lor agglutination also.
The predpitanort test may be Carried out as e]ther a
qualitative or quantitative test. It is veiy sensitive in
tile detection of antigens and as little as 1 ggot protein
can he detectedbv precipitation tests. It Therefore finds
forensic iipplication in the identification ofblood and
seminal stains, and in testing for food adulterants.
Precipitation is nclutivelv less sensitise for the detection
of antibodies.
The following types of precipitation and
flocculation tests are in common use;
Tb]Shthe simplest type of precipitation
test, consists of layering the antigen solution over a
column of antiserum in a narrow tube. A precipitate
forms at the junction of the two Liquids. Ring ces^
have only a few clinical applications now. Examples
are AsCotis thermoprecipitin test and the grouping
of streptococci by the T.arccfidd technique.
When a drop each of the antigen and
antiserum are placed on a slide Jnd inixuL by
shaking, floceules appear, The V D RL rest for
syphilis is an example of slide flocculation..
The Kahn test for syphilis is an
example of a tube flocculation test. A quantitative
tube flocculation rest is employed for Ilie
stancLireEisatidTi of trains and toxoids. Serial dilutions
of the toxin/toxoid arc added to the tubes containing
a fixed quantity of the antitoxin. The amount of
toxin or toxoid that flocculates optimally with ore
unit of the antitoxin is defined as an I i dose.
C o p y rig h te d m aterial
 ; 7. ::: cl

(D

o o o o
0 c
_ _ - - - _,J --- ------------------ . . . .. ,
- |G j :. •' ,'Lti
hi-ttK ritaMPttSM (Qs&ls^—Fiiiiihcrpiftl RidW Hutu It dWustan In tea c,te
fMWHMi ff Wtatlty (A), panto S3 I^V lifU IKS . rSL"
■w,
■
-■ - ■■' H ^miserum s a t m

precipitation formed at ihc advancing front of the

There arc several advantage* in allowing antigen, and in dissolved as the concentration of
pncci pita non to occur in t gjd rather than in a Liquid antigen ic the site increases due m diffusion, I he
medium. The reaction is visible AS A distinct band number nf hands indicates the number of different
of precipitation, which is stable and tan be stained antigens present.
for preservation* if necessary- A* each antigen " 2, D o u b le diflfusinrt m o n e dim en sion (O .iklr i —
antibody reaction gives rise to a line of precipitation* Ftiltharpe procedure): Mere, rhe antibody is
the number of different antigens L:i the reacting ineorpn rated in gel* above which is placed a
mixture can be readily observed- Iimmunodiffusion column of pla ii .M i He antij ii is iyi ■■ i t
also indicates identity* cross-reaction and nonidenrity top of this, t : irigen d mtibodj :i ■■■■:
hetween different antigens. toward* each other through ibe intervening
Immunodiffusion is usually performed in a soft column ' |- ■m aga: .md lorn t a band iifpret , ■i1 1 1 .
(1%) agar or agarose gel. Different modifications where i Iil-v meet at optimui import ..:
of the test are available; 5, Single d ifliis h n fn tiw dnnem ions i Radial
i. S in g le diffusion in one dimcjjfiuri (O udin j rrurpivf.'i >>j'j f i*jj*i.-m ■'■ H ere the antiserum is
procedure): "Hie antibody LS incorporated in agar incorporated in agar gel ■.- nn d ■-n a flat surface
gel in a test tube and the antigen solution is layered (slide Or Petri dish). The untiu; ■■ is added to the
over ir. The antigen diffuses downward through wells cut on the surface of te gel. It diffuses
the agar gel, forming a line of precipitation that radially from the well and font] ring-shaped
appears to move downwards- This is due to die bands of precipitation h llot concentric illy

C o p y rig h te d m aterial
 < Antigen— Antibody reactions *
around the well. The diameter of the halo gives
m estimate of the concentration of the anrigen.
This method has been employed for the estimation
of the iinmunugiubtjjin classes in sera and fur
screening sera lor antibodies to influenza viruses,,
among others.
4. D ouble diffusion in two dim ensions
(O ochterlonv procedure): This is the
'.mmunodiffusion method most widely employed
and helps To compare different antigens and
antisera directly. Agar gel is poured on a slide and
wells arc cut using a template. The anberum is
placed in the central well and different antigens
in the surrounding wells. If Two adiaccnt antigens
are identical, the lines of precipitate formed hy
them will fuse. If they are unrelated, the lines
will cross each other- Cross-reaction or partial
identity is indicated by spur formation (Fig. 13.3].
A special variety of double diffusion in two
dimensions is the Elek test for toxigenieity in
diphtheri.il bacilii. When diphtheria bacilli, are
streaked at rl^ht angles to a filter paper strip
carrying the antitoxin implanted on a plate of
suitable medium, arrowhead shaped lines of
precipitation appear on incubation, if the bacillus
is tuxigenic,
5. Immunoelectrophoresis: The resolving power
of immunodiffusion was greatly enhanced when
G ra b ar and W illia m s deviled the technique o f
immunoelectrophorcsis. This involves the
clcctiophorctie separation of a composite antigen
(such as serum ) into its constituent proteins,
followed by immunodiffusion against its antiserum,
resulting in separaic precipitin lions, inuieating
reaction between each individual protein with its
a n tib o d y . This enables id e n t if ic a t io n and
approximate quantitation o f the various proteins
present in the scrum . The technique is performed
on agar or agarose gel on a slide, with an antigen
well and an anlibody trough cut o n it. The test
se ru m is placed in the antigen well and
elecrrophoteaed for about an hour. A n tib o d y
against human serum is then placed in the trough
97
and diffusion allowed to proceed for 13-24 hours.
The multing precipitin Lines can be photographed
and the slides dried, stained and preserved for
record. Over 30 dilforcnt proteins cun be identified
by this method in human serum. This is useful
for testing for normal and abnormal proteins in
serum and urine (Fig. 13.4).
II le t Lrui m mun Dili flu s ion: The development
of precipitin lines can be speeded up by electrically
driving the ,irnigen and amibody. Varlam, method1-,
have been described combining electrophoresis
with diffusion. O f these, one-dimensional double
elcctroim mu nodiffusion (counterim m uno-
elcctrpphnrc'i-.) and one-dimensional single
electroimmunodilTusnin (rocket electrophoresis)
are used frequently lit the clin ical laboratory.
J,Counteri.,]TmLrf]cie/ccrrophore‘ii5 fCIE, counter*
current immunoelectrophoresjsfc This involves
simultaneous electrophoresis uf the antigen and
antibody mgel in opposite direciions resulting in
precipitation at a point between them (Fig. 13.5).
This method produces visible precipitation lines
within thirty minutes and is ten times mote
sensitive than the standard double diffusion
techniques. The clinical applications are for
detecting various antigens such as alph.Lteroprocein
i n scrum and spec ihe anti gens of cryptocoeous and
meningococcus in the cerebrospinal fluid,
J . Or?f JjrjJiT^Ki-Ji.i.1Sjqgfe eicctzv u in n isu o d ith iy iiu ?
(rocket electrophoresis): The mai ri application of
this technique Is for quantitative estimation of
antigens. The antiserum to the antigen TO be
quantitated is incorporated ill agarose and gelled
an the glass slide. The antigen, In increasing
concentrations, is placed in wells punched in the
set gd. The antigen is then eleecrophoresed into
the amibody containing agarose (Fig. 13.6). The
pattern of imu juuoprecipitation resembles a rocket
and hence the name.
A variant of tins is Laurell's two-dimensional
electrophoresis. In tins technique, the antigen
mixture is first electropharetLcally separated in a
direction perpendicular to that o f the final rocket
C o p y rig h te d m aterial
 stage. Ry tKas. method one cart quantitate each of
several antigens in a mature (Fig. 13,7),

When a particulate antigen is mixed with its antibody

in the presence of electrolytes at a suitable temperature
and p llhthe panicles are dummied or agglutinated,
Agglutination is more sensitive than prpt'i pi ration for
the dcinttKMiof antibodies, 1 “he name pri nc iplea govern
agglutination and precipitation. Agglutination occurs
optimally when antigens and antibodies react in
equivalent proportions. The /cine phenomenon mav
he seen when either an antibody or an antigen is in
excess, "Incomplete’or 'monovalent' antibixiLCH do not
cause agglutination, though they combine with the
antigen. They may act as ‘blocking1 antibodies,
inhibiting agglutination by rhe complete antibody
added subsequently,

t:. vl . .... ,. , ■:■■.}

I:.: i;,3 r iD 7 j
l >1 - When 3 drop of the
appropriate antiserum is added to a smooth, uniform
suspension o f a paniculate antigen in a drop of saline
on a slide or tile I agglu ti nation tj kes place. A positive
result is indicated by the clumping together of the
particles and the clearing of the drop. The reaction
is tacilitated by mixing the antigen and the
anflscrum wi th a loop or bv gen tlv nic ki ng tlie slide,
[depending on the titne of the sc mm. agglutination
may occur instantly or wirhln seconds. Clumping
occurring after a minute may be due lo drying Ol
the fluid and should be disregarded. It is: essential
to have on the same slide a control consisting of
the antigen suspension in sa!ineh without the
antiserum, to ensure that the antigen is not
autoaggluti [table. Agglutination is usually visible
to the naked eye hut may sometimes require
confirmation under the microscope, Slide
agglutination is a routine procedure for rhe
identification of many bacterial iso lutes from
clinical specimens. It is also the method used for
blood grouping and cross- matching.

C o p y rig h te d m aterial
 antigens are used. The 'IT or the flagellar antigen
on combining With ir> jn:LhoiU lorni'. l.irgL, kh>sc,
fluffy clumps resembling mu-ps of eottonwooL '['be
Electrophoretic currant O or so m,iric antigei f imis tight, eonnjiaeldtp* v$i [-
re semi ding fh.dk powder. Agglutinated bacilli
spread OCt in a djSClike pattern at die bottom of the
m hev
T h e tube agglutination resT for brucellosis may
© = I= © lie complicated hi' llie proaonc phenomenon and
th e presence o f 'b lo c k in g ’ an tib o d ies. Several
dilutions o frh e scrum should be tested to prevent
false negative results due to pmaone. Incom plete
or hijh'kinjr antibodies may he lJerected hi' doing
The tesr in hypertonic (S4^ ' saline of albumin saline*
, - Hr
n . ,'. , -rJc-=■!■a-B
rm '■-1 1
; ■ - 17 _ f
,■, rl “ -.’t.-mti
r -m w- jjg B i E V J - ■=■ — ^

,Tl ifitibcfj ft| frivsn ipgetbfn if 3P
Gitrtrnl stir] L pr
This is a standard
quantitative method fot the measurement of
antibodies When j fixed volume ot a paniculate
antigen suspension is added To an equal volume of
serial dilutions of an antiserum in test tubes, the
agglutination litre of the serum can be estimated-
Tubc agglutination is routinely employed f<W the
serological diagnosis of typhoid, brucellosis and
typhus fewer.
In the Widal test used in typhoid* two types of
or more- rrSiahli by rive anrigiohulin (CiNvrnbh) test,
fl 1sn# Iflf lit, Jfhc Weil-Fch* reaction for sciodijignosT of
typhus fevers is a lieisitvpEiile agglutination test and
is based oil Ihr sharing of a com til on antigen
between typhus richelts-i-ie and some strains of
protcuc bacilli.. Another example of the htterophile
agglutination lest is the Strc/itucflceuf MG
agglutination rest for die diagnosis of primary
atypical pneumonia.
Examples of agglutination tests using red cells
as antigens are the Paul Bonne I test .md the cold
agglutination text. T h e form er i- based on the
presence ot -nheL'ji cell aj^jlutinin- in I he s-era qf
in fe ctio u s m o n o n u cleo sis p atien t^ w hich are
adsorbed by OX red cells- but not by gUirke.H pig kidneV
e x tri.r. J in- . -.1.1 agglutination iect po&iiive in
mwopLwna] (primary atypical} pneumonia. The
patient's sera agglutinate human O group
erythrocytes at 4 <,C , the agglutination heing
reversible at 37 T ,
antiglobulin test was devised by C oombi* Mourant
and Race (1945) for the detection of anri-Rh
antibodies that do not agglutinate- fth positive
erythrocytes in saline. When sera containing
incomplete anii-Rh antibodies are mixed with Kh
positive red cells, the antibody globulin coats the
surface of the crvthrncytcs, though they are not
agglutinated. When such erythrocytes coated with

C o p y rig h te d m aterial
 i I ' I1 f -
. . . i _____ : .
the anttbody globulin arc washed free of all
unattached protein and treated wirh a rabbit
antiserum against human gammaglobulin
(antiglobulin or Goomb> ^crum), the cells ,irt
igglu [mated. This is the principle of the antiglobulin
test (Fig. 13,6).
T h e C fjoinbs test muv he direct or indirect. In
the direct Cwnqbs test, the sensitisation of the
erythrocytes with mcortplere antibodies takes place
in vim, js in the he mol yin: disease or the newborn
due co Kh incompatibility. When the red ceils uf
e ty th ro bias Lot ic EEiiants ate washed free ot
unattached prnrem and then mixed w iih :l drop of
Coombs serum , agglutination results. T h e direct
C o o m b t [esl ]s uitEd lU p t iv t in hemulvtic disease
due to A B O incm iiparihilitv.
In the indirect Coombs test, sensitisation of red
cells with the antihody globulin is performed in
vitro. Originally employed for detection of anti-
Rh antibodies, the Coombs tes1 es useful for
demonstrating any type of incomplete or
nonagglutinating antibody, as, for example, in
brucellosis-
- i : The only
difference between rhe requirements for the
precipitation and agglutination tests is rhe physical
nature Ot lire antigen. Bv attaching soluble antigens
— - ’-J :: - ;■ ' -.7 ‘
to the surface of carrier parcu lc . it is possible 10
convert precipitation rests into inaiiun tests,
which are more convenient and ■in! sensitive for
the detection ot antibodies. Sin tests arc known
as iusurt jugfiitvMtiMi rests.
T h e Cufllxnojdy used carrier parli; are red
cells, latex particles or bentonite. J Inn u or sheep
erythrocytes adsorb a vitietj o f iiiitigem
Polysaccharide antigens m ay be i | ■■ : k \L :■■. -I.rp i.
mixing with the cells. For idsor^ni 11 of protein
antigens, tanned red cells are .-.i
A special typeof passive ir 1 i.Lgi lutimidati test
is the Rose Waaler test. In iniatoii arthritis,
an aulLunribody (RA factor) ■■■ in the SCfUITl,
which acr*. .ls an fcntibody to ;....... u- ......Li-11. The
RA factor is able to lul I . :•■ il.. Cetls Coated
with globulins, lilt: antigen used for the resl is a
suspension o f sheep erythrocytes seinitistd with a
sub agglutinating dose of rabbit amishccp
erythrocyte antibody (amboceptor).
FVlIystyrene 11 RX, which can he manufactured
as uniform spherical particles, O.S-t m in diameter,
cat; adsorb several types of antigens;. Latex
agglutination tests (latex fixation tests) are widely
empteyed in the di nica I laboratory tor the detect!on
of ASO, CRP, RA factor, HCG 5nd many other
antigens.
iPY rkihted m aterial
 J3asHivc ugglu t ina cion tests arc very sensitive and
yield high titter but may give false positive results.
When instead of the antigen, the antibody is
adsorbed to farrier particles in tests for estimation
of antigens, the technique is known, as reversed
passive agglutination.
c o m pu m en t fixation i f f t fe n )
Complement takes part in many immunological
reactions and is absorbed during the combination of
antigens with their :intibt>dieH. In the presence tjf the
appropriate antibodies, complement lyses eiyrthrexytes,
kills and, ill some cases, lyses bacteria, immobilises
motile organisms, promotes phagocytosis and immune
adherence and contributes to tissue damage in certain
types of hvpersensitivity.
T h e ability' o f antigen antibody complexes to
fix' complement is made use of in the complement
fixation test (C FT ). Thin is a vety versatile and
sensitive test, applicable with various types of
antigens and antibodies and capable of detecting as
little as 0.04 mg of antibody nitrogen and 0.1 mg
of antigen. C F T is a complex procedure consisting
of two steps and five reagents—antigen, antibody,
complement, sheep erythrocytes and amboceptor
{rabbit antibody to sheep red cells). Each of these
reagents has to be separately standardised.
The antigen may be soluble or particulate. The
antiserum should be heated at 5b aC [inactivated)
for half an hour before the test to destroy any
complement activity the semm may have and also
ro remove some nonspecific inhibitor* o f
complement present in some sera
[anticomplementary activity). The source of
C o p y rig h te d m aterial
complement is guinea pip serum. As complement
iftiviiv is K«at labile, the serum should be freshly
drawn, or preserved either in the lyophllised or
frozen stare or with special preservatives, as in
Richardson's method. The guinea pig serum should
be titrated for complement activity. Otic unit or
minimum hemolytic dose (MHD) of complement
is defined as the highest dilution of the guinea pig
serum that lyses one unit volume of washed sheep
erythrocytes in the presence of excess hemolysin
(amboceptor) within a fixed time (usually 30 or 60
minutes) at a fixed temperature (3? "C). The
amboceptor should he titrated for hemolytic activity.
One MHD of amboceptor is defined as the least
amount (or highest dilution) of the inactivated
amboceptor that lyses one unit volume of washed
sheep erythrocytes in the presence of excess
complement within a fixed Time (usually 30 or 60
minutes) at a fixed temperature (37 "CJ.The diluent
used for the titrations and for C F T is physiological
saline with added calcium and magnesium ions.
The classical example of C R T is the
Wasscrmann reaction, formerly the routine method
for the senodiagnosis of syphilis. The test consists
of two steps. In the first, the inactivated serum of
the patient is incubated at 37 ^'C for one hour with
the Wasscrmann antigen and a fixed amount (two
units) of guinea pig complement. If the serum
contains syphilitic antibody the complement will
be utilised during the antigen antibody- interaction.
If the serum docs not contain the antibody, no
antigen-antibody reaction occurs and the
Lumplemenl will therefore be left intact. Testing
for complement in the postincubation mixture will
thus indicate whether the scrum had antibodies or
not. This constitutes the second step in the test and
consists of adding sensitised cells (sheep
ciythrocytes coated with 4 M HD hemolysin), and
incubating at 37 E'C for 30 minutes. Lysis of the
erythrocytes indicates that complement was not
fixed in the first step and, therefore, the scrum did
not have the antibody (negative CFT). Absence of
erythrocyte lysis indicates that the complement was
used up in the first step and, therefore, the serum
contained the antibody (positive C FT ) (Fig. 13-9).
Appropriate controls should be used, including
the following: antigen and serum controls to ensure
that they are not anticomplemcntary, complement
control ro ensure that the desired amount of
complement is added, ami cell control to see that
sensitised erythrocytes do not undergo Iitls in the
absence o f complement.
Certain avian (for example, duck, turkey, pamot) and
mammalian (for example, horse, cat) sera do not fix
guinea pig complement. When such sera are to be
tested, the indirect complement fixation nest may be
employed. Here the test is set up in duplicate and
alter the first step, the standard antiserum known to
fix complement is added to one set. If the test serum
contained antibody, the antigen would have been used
up in the first step and thcretore the standard antiserum
added subsequently would not be able to fix
complement, Tlueiefore, in the indirect test, hemolysis
indicates a positive result.
For systems which do not fix guinea pig
complement, an alternative method is the
opngluliniiting com plem ent absorption test. This
uses horse complement which is nonhemolytic. The
indicator system LH Sensitised sheep erythrocytes
mixed with bovine serum, ftovine senim contains a
beta globulin component called congiutinin, which
acts as antibody to complement. Therefore,
congiutinin causes agglutination of sensitised sheep
erythrocytes (conglutination) if they have
combined with complement. If the horse
complement had been used up by the antigen-
aittjjoody interaction in the first step, agglutination
of sensitised celIs will not occur.
When some bacteria (for
example, Fjfbrj'o choJerae, Treponema pallidum)
react with the specific antibody in the presence o f
complement and particulate materials such as
erythrocytes or platelets, the bacteria are aggregated
C o p y rig h te d m aterial
 4 reactions ►
and adhere to the tells, This is known as immune
adherence. The j mmobiliaa tfon t e s t is arturlier
complement dependent ractiofi, Tin the Treponema
pallidum immobiHsMtioa test, a highly specific test
formerly considered the 'gold standard' for the
semdiagruttis of syphilis, the test serum is muted
with a live motile suspension of T. pallidum in the
presence of complement. On incubation, the specific
antibody inhibits the motility of treponemes.
Cvtttlytic [?r cytooidil tests are also complement
dependent. When a suitable live bacterium, such as
the cholera vibrio, is inoted with its antibody in die
presence qf complement, the banterium Ls killed and
Eyscd. This forms the basis of the vihriocidd antibody
test for the measurement of anticholcra antibodies.
NEUTRAUSAT10N TESTS
Neutralisation
of viruses by their antibodies can be demonstrated
in various systems. Neutralisation of bacteriophages
can be demonstrated by the plaque inhibition test.
When bacteriophages are seeded in appropriate
dilution on lawn cultures of susceptible bacteria,
plaques of lysLt are produced. Specific am iphage
senun inhibits plaque formation. Neutralisation of
a nimal viruses can be demonstrated in three systems
- animals, eggs and tissue culture.
l. ANTIGEN + TEST SERL'M
{CONTAINS ANTIBODY)
+ COMPLEMENT
+ H E M O L Y T IC S Y S T E M
II A N T IG E N + T E S T S E R U M
(C O N T A IN S N O A N T IB O D Y )
+ CO M PLEM ENT
+ H E M O L Y T IC S Y S T E M
103
bacterial exOtcurins are
good antigens and induce the formation oi
neutralising antibodies (antitoxins) which are
important clinically, in protection against anil
recovery from diseases such as diphtheria and
tetanus. The toxicity of endotoxins is not neutralised
by antisera.
Toxin neutralisation ear be tested in vivo or in
vitro. Neutralisation tests in animals consist of
injecting toorin-untitoxin mixtures and estimating
the least amount of antitoxin that prevents death or
disease in the animals. With the diphtheria toxin,
which in smalt doses causes a Cutaneous reaction,
neutralisation tests can he done on rabbit skin. Tlie
Schick test is based on the ability of circulating
antitoxin to neutralise the diphtheria toxin given
intridermally, and indicates immunity or
susceptibility to the disease. Toxin neutralisation
in vitro dc pends- on the inhibit ion of some
demonstrable toxic effect. An example is the
ami streptolysin O test, in which antitoxin present
in patient sera neutralii.es the hemolytic activity of
the streptococcal O hemolysin,
OPSOMSATKM
The name Jop5nniri was originally given by Wright
(1903) to a heat labile substance present in fresh
- C O M P L E M E N T H IX E D
RESULT - NO HHMOi .YSlS
POSITIVE CF TEST
COMPLEMENT NOT FIXED
R E S U L T -H E M O L Y S IS
N E G A T IV E C F T E S T
C o p y rig h te d m aterial
104 * TfrKibook flf Microbiology s
normal scri, which facilitated phagocytosis, 1 bi>
factor was subsequently identified as complement.
A hcatstahlc scrum factor with similar aclnity was
called 'hactermirupin'. This appears to be a speLtfic
antibody. The term opsonin is now generally used
to refer to both these factors. Wright used the
'opsonic index' to study the progress of resistance
during the course of diseases. The opsonic index
was defined as the ratio of the phagocytic activity
of the patient’s blood ft>r a given bacterium, to the
phagocytic activity of blood from a normal
individual- It was measured by incubating fresh
citrated blood with the bacterial suspension at
37 "C for 15 minutes and estimating the avenge
number o f phag^tcytosed bacteria per polymorpho
nuckar leucocyte (phagocytic index) from stained
blood films.
IMMUNOFLUORESCENCE
Fluorescence k the property of absorbing light rays
of one particular wavelength and emitting rays with
a different wavelength- Fluorescent dyes show up
brightly under ultraviolet light as they convert
ultraviolet into visible light. Coons and his
colleagues (1942) showed that fluorescent dyes can
be conjugated to antibodies and that such labelled’
antibodies can be used to locate and idemily
antigens in tissues. This ‘fluorescent antibody’ or
immunofluorescence technique has several
diagnostic and research applications.
In irs simplest forms (direct immuno­
fluorescence t e s t ) , it can be used for th e
identification ofbactervi, viruses or other anti.Ljens,
using the specific antiserum Labelled with a
fluorescent dye. For exam ple, direct immuno­
fluorescence is routinely used as a sensitive method
of diagnosing rabies, by detection of the rabies virus
antigens in brain smears. A disadvantage of this
method, is that separate fluorescent conjugates have
to be prepared against each antigen to be tested.
The "indirect Lmmunofluoresuence teit’ overcomes
this difficulty by using an antiglobulin fluorescent
conjugate. An sample is the fluorescent treponemal
antibody test for the diagnosis of syphilis, i [ere, a
drop of the rest serum is placed on a smear of T
pillidum on a slide and after incubation, the slide
k washed well to remove all free serum, leaving
behind only amibody globulin, if present, coated
on the surface of the tTepcmcmes.The smear k then
treated with a fluorescent labelled antiserum to
human gammaglobulin- The fluorescent conjugate
reacts with antibody globuiin bound to the
creponemes. After washing away all the unbound
fluorescent conjugate, when the slide is examined
under ultraviolet illumination, if the tesr Is positive
the tneponemes will he seen .is bri i^bt obj ects agai list
a dark background. If the serum does not have
aim treponemal antibody, there will be no globulin
eoariog nn the irrpoticmci and therefore they will
not take on the fluorescent conjugates, A jingle
anti human globulin fluorescent conjugate can be
employed for detecting human antibody to any
antigen iFig. 13.10).
Fluorescent dyes may also be conjugated with
complement. Labelled complement is a versatile
tool and can be employed for the detection of
antigen or antibody. Antigens also take fluorescent
labelling but not as well as anti bodies do. For
detection iif antibodies by imniunofluorciceri-'e, the
sandH rLb techimyUe can be employed.The autihodv
is first allowed IO react With unlabelled antigen,
which is then treated with fluorescent labelled
antibody A sandwich is thus formed the antigen
being in the middle and libelled and unlabelled
antibodies on euher side.
The fluorescent dyes commonly used are
fluorescein isothiocynare and lissamine rhodamine,
exhibit ing blue-green and orange-red fluorescence,
respectively. By combining the specificity of
serology with the localising capacity of histology,
immunofluorescence helps in the visualisation of
antigen-antibody reactions :in situ. It I-; thus an
immunohistochemical technique. The major
disadvantage of the technique i*. the frequent
occurrence of nonspecific fluorescence in tissues
and other materials.
C o p y rig h te d m aterial
 i Arttgtn— Antibody m d lo m *

RADtOtMNUMQASSAY (RM) the hinder. The first reaction ot this type was

Besides fluureseent J v t m a n y other distinctive radioim m unoassay ;R ] A ) described hv Rerson and

'labels' nisei i,-jn he conjugated to antigens and Yallenv i[] 1959. RJA J>CTirai t ^ the measurement of

antibodies, Tile most com m only used Libels arc
radioisotopes -nul emtyiues. A variety of tests h iv e
been devised for the measurement o f antigens and
antibodies using such labelled reactants. T h e term
b in d c r-lig tn d -iS $ a y has been u sed for th ese
re a c tio n s . T h e su b sta n ce ( a n tig e n ) w hose
eom xnt ration is rn he denTiriined is lenned the
flpUt/lTe tjt litfdiu i. J lie binding protein i ordinarily,
the antibody) w hich binds, to the ligand is called
analyses uptn piengmm ( 1 0 11 gl quantities. R IA
and it* modifications have versatile application* in
various areas o f biology and m edic ire , including
rhe quantitnrion of hormones, drugs„ tumour
markeiSj, IgE ami vara! antigens. T h e importance
o f RIA was acknowledged when the NohcL fr it e
was, awarded ns Yullnw for its discovery in 1977.
RIA is a Coinpetilive binding asrav in which
Fixed amounts of antibody and radiolabelled antigen

+
$

Fig 1&1Q lm iiunvftof*w *n»L 1. D trv c tM : .ftriftip1 }\> It ndxvd with lbp;n^^tnf«oidf psd antibody (H).
H it tnHgt ii wWbody complev It ftto n w H it (Cl= R Imflnpet t ill; A ntigtt (A) t i mfawd <nHh g u tte r
(B]iThi ntdgan^mSiMidr compfc* (C) f m is I with 1hioi«icMl eonfuguni! simin. |D) TUto
106 * Texlbook of Microbiology
react '.ii the presence n f h la b e lle d antigen- Jfh c
labelled and Urtlabelled antigens compete Tut [he
limited binding sites on the antibody. This
camper it ion is determined by the lewd of the
unlibelled (rest) antigen present in the reacting
system. Alter the reaction, the antii;en is separated
intio Tree* and “bound' fra*' riims and their radioa* rive
counts measured. The concentration of the test
antigen can be calculated from the ralio o f the
bound and total antigen labels^ using a standard
dose response curve.
Enr anv reacting system, the standard dose
response or calibrating curve has to be prepared
first- This is done by running the reaction with
fixed amounts ■■t antilH>dy and labelled antigen, and
varying known amounts of unlabelled antigen. The
ratios [)1 Ik uiiul : total labels i B : 7' 15.(1 0 ) plotted
against the analyte concentrations give the standard
calibration curve. The concentration of antigen
in the test sample is computed from the R : T ratio
of the test by interpolation from the calibration
curve.
ENZYME IMMUNOASSAYS (ELA)
Enzyme labelled conjugates were first introduced
in 19*6 for localisation of antigens in ti-sucs, as an
alternative to fluorescent conjugates. In 1971,
enzyme labelled antigens and antibodies were
developed as serological reagents for the assay of
antibodies and an Ligens. Thei r versarilii 1 , sen--! t ivity,
rimpi icity, economy and absence of radiation h azard
have made ElAs the most widely used procedure
in clinical serology. The availability of test kits
and facility for automation have added ro their
popularity.
1 he term ejlzrroe imnmnoitssii^EIA) includes
all assays ha*ed on the measurement of enzyme
labelled antigen, hapten or antibody. EIAs are of
two basic types - homogeneous and heterogeneous.
In homogeneous E1A, there- is no need to separate
the bound and free fractions so that the test can be
completed in one step, with all reagents added
simultaneously, Thu type of El A can be used only
*
for assay of hapten* such as drugs and not for
microbial antigens arid antibodies. An cx.imple of
homogeneous E l A is enzyme m ultiplied
j'liimujmas&ty rec/urigue (EM IT), which is a simple
assay method Tor small molecule drugs such as
opiates, cocaine, barbiturates or amphetamine in
serum.
Heitir^eno: El A requires the reparation of
tlic lree and hound frac tior i>tic her Iy ce 1itri lugation
or by absorption on solid surfaces and washing, it
is therefore a muliisrep procedure, with reagent*
added sequenti:J]y. 7*hc major type of heterogeneous
E l A is Enzyme Linked Immunosorbent Assay
(ELISA). '
Enzyme Linked immun&snrbent Assays
(ELISA).
ELISA is so named because the technique
involves the use of an artTmcjflusorbenr - an
absorb mg mare i u l specific for otic of the
components of the reaction, the antigen or antibody.
This may he particulate, for example cellulose or
agarose - or • solid phase such as polystyrene,
polyvinyl or polycarbonate tubes or mtcrowells -
or membranes or discs of polyacrylamide, paper or
plastic, ELISA is usually done using 96-well
Tuic-rocitre plate* *uifable for automation. The
principle of the test can be illustrated by outlining
1re applies rion for the detection of rotavirus antigen
in feces.
The wells of a microti be plate arc coated with
goat anti rotavirus antibody. After thorough wasliing,
the fecal samples to be tested are added and
incubated overnight at 4 ^ or for two hours at
37 SC. SuLiable positive and negative controls are
also SCT Up. The wells arc washed and guinea pig
antirotavi rus anriserum, labelled with alkaline
phosphatase, added and incubated at 37 °C for one
hour. After washing, a suitable substrate
■;paranitrophenyi phosphate) is added and held at
room temperature till the positive controls show
the development of a yellow colour. The
phosphatase engine splits the suhstrate to yield EL
yellow compound.
C o p y rig h te d m aterial
 If the test sample contains rotavirus, ir is feted
to the antibody coating the wells, When the enzyme
Labelled antibody is added subsequently, it is in turn
fixed. The presence of residual enzyme activity,
indicated by the development of yellow colour,
(here tore denotes a positive test (Fig. 13.11). If the
sample is negative, there is no significant colour
change. An ELISA reader provides quantitative
colour recordings.
The detection of antibody by E l. ISA can be
illustrated by the anti-HIV antibody test. Purified
inactivated HIV antigen is adsorbed onto microassay
plate wells. Test serum diluted in buffer is added to
the well and Incubated at 37 UC for 10 minutes.
rfhc wclE is then thoroughly washed. If the serum
contains anti-HIV antibody, it will form a stable
complex with the 1 1IV antigen on the plate. A goat
anti human immunoglobulin antibody conjugated
with hoiae radish peroxidase enzyme is added and
incubated for 30 minutes. After thorough washing,
the substrate O-phtnylene diamine dihydroehlorLde
is added and after 30 minutes, the colour that
develops is read using a microassay plate reader.
Positive and negative controls should invariably he
used with test seia (Fig. 13.12).
The examples described above are of simple
nonoo/nj3cn"fjVe .samJu-frb ELISA. The test can be
made more specific by miking scrum antibody and
enzyme labelled antibody compete for the binding
sites on the antigen (rompenfiVe ELISA). Tests
C o p y rig h te d m aterial
for specific immunoglobulin classes (tor example,
IgM spiit'jtic ELISA) arc also available Coptine
E L ISA and rmunj/JonietTj'c tests arc m n marc
specific.
Severn! variations of the ELISA technique have
been developed ro proride simple diagnostic teds,
including the card and dipstick methods suitable
for clinical laboratory' and bedside applications.
A sim ple m odification o f E l . IS A w hich has
found w ide application for testing one or a few
samples of sera af a rime is the cylinder or GMseffe
ELJSA. Here each specimen is tested in a separate
dis-posablf cassette. T h e test is rapid, taking onlv
about !() m i notes as compared with the 2 -4 hours
taken for mieropiatc EvLISA. There is no need for
micrnplate washers or readers. T h e result is read
visually. Inbuilt positive and negative controls are
usually provided for validation of the test procedure.
An example of cassette E L IS A is rbc one used
for the detection of HIV type 1 ;ind 2 antibodics.
SpeoBc type 1 and 2 antigens are immobilised at
separate tbred ‘■ires on the nitrocellulose nim b nnt
ml the cassciie. Test serum is added on the
m embrane and allowed to tiller into absorbent
m aterial placed below it in the cassette hast-
Amibody. if present in the scrum will bind to the
appropriate antigen. After washing to remove
unbound antibody, enzyme lube! led a mi bum an
immunoglobulin antilwidy is added. After additional
washing to remove unbound conjugate, a substrate
yielding a coloured product is added. A positive
result is indicated by a coloured spot developing at
the site of the antigen against which antibody is
present in the strum. Human immunoglobulin
immobilised af a spot on the membrane acts as a
control for the test procedure, as shown by the
development of colour at the site.
Chemihi mirtescence ttfifcft (o a chemical reaction
emitting energy in the form of light, just as
radioactive conjugates ate employed in RJA
fluorescent eojugarcs in IFA and enzymes in ELISA.
chemiluminescent compounds (such as luminol or
acrid ini um esters) are used in CLIA as the label to
provide the signal during the antigen-antibody
reaction. The signal (light) can be amplified,
measured and the concentration nf th e analyte
calculated. The method has been hilly automated
and is being increasingly user! in laboratories where
rlx volume of work is large
Im m unoclcctroblot or (clectroim m ij noblot)
Techniques combine the sensitivity o f enzyme
im m unoassay w ith m uch greater spec i fie i fy. T h e
technique is a combination o f three separate
procedures: (a) separation o f Ligand-antigen
components by polyacrylamide gd. electrophoresis'
[bl blotting ol the electnopboncscd ligand fraction
on nitrocellulose inemlwanc strips; and (c) enzyme
immunoassay (or radioimmunoassay) to (1) detect
antibody in rest sera against the various ligand
fraction bands; or (2) probe with known antisera
against specific antigen bands. The Western Blot
rest, considered die definitive test for the
serculiagnosis o f HIV infection, is an example o f
the immunoclectToblor technique (see chapter 62
for details).
A one-step qualitative immunocbromatographic
technique has found wide application in
xrodiagnosia due to ifs simplicity, economy and
reliability. A description o f its use for HEsAg
detection illustrates the method. The test system 15
a small cassette containing a membrane
impregnated with anti-Hb&Ag antibody-colloidal
gold dye conjugate- The membrane is exposed at
three windows on the eastern;, The rest serum is
dropped into the first window. A s the serum travels
upstream by capillary action, a coloured band
appears at the second window (test site) if the serum
contains HbsAg, due to the formation of a HbaAg-
antibody conjugate compIeK.This is the positive
reaction. Absence of m coloured band at the test
C o p y rig h te d m aterial
site indicates a negative reaction. Simultaneously a
coloured hand should appear in every ease at the
third window, w hich forms an inbuilt control, in
the absence o f w hich the te^C is invalid. The tent
claimed to be nearly as sensitive and specific as
ElA tests.
W h e n v ira l
particles mixed with specific antisera ate observed
under the electron microscope, they are seen to be
clumped. T h is finds application in. rhe study o f some
viruses Sudl as the hepatitis A virus and the viruses
causing diarrhea.
Ferritin (an electron -
dense substance from horse spleen) can be
conjugated with antibody,, and such labelled
lh antibody reacting with an antigen can be visualised
under the electron microscope.
Some stable enzymes*
such as pCrtuudase, cun Ijc conjugated with antilKjdjes,
Tissue sections carrying the cocres|iondirtg antigens
arc treated wirh peroxidase labelled antisera. The
peroxidase bound to the antigen car he Yurutaliwd under
the electron microscope, by mictohistnchemical
iviL-rtuvis. Some other crtzynncs, such as glucose oodrfase,
phosphatases mod tyrosinase, may also be itidudjedi in
imnumoenzyme tests.

Hnlnwri A Ft al. 1S9]. Marumi of CliniciJ .MjVnjfnprJ^gyLW^ihLngtyr. DC: Amerkan Surety of Microbiology.
Hudson L And FC Hay 1999. firaL-n'cid hnmunalogy^ > ' cdn. Oxford: Blackwell.
Kennedy CM. and SJ Challacombe 1959. ELISA and Other Solid Phnic lnvnuiio**ap&. NtwYole John VVilry.
Knse NR ec al. 1986. Mamiil oFClinii-il fmjuurrok^y. 3rAodn. Washington OCt American Society for MifciobidoKy.
Stiles DP and AE Ten 1991. B i i i e a n d O lim c a lIm in iiT ttittT g y ^ 7* fdn. Norwalk Appleton-l-an^c.

C o p y rig h te d m aterial
 The Complement System
The term ‘complement' (C) refers to .l system of
factors which occur in norma] serum and are
activated characteristically by antigen-antibody
interaction and subsequently mediate a number of
biologically significant consequences.
Towards the end of the nineteenth century, lt
was noticed that bactericidalh bacteriolytic and
hemolytic actions of the appropriate antibodies
requi red the parr ic ipari on of a heat labile component
present in the normal sera of human beings and
animals. Buchner :l!?t89) was the fust to observe
that the bactericidal effect of serum was destroyed
by heating at 55 aC (or one hour. Pftelfer (1B94)
discovered that cholera vibrios. were lysed when
injected intraperi toneallv into speri tic ally
immunised guinea pigs (bacteriolysis in vivo or
Ffieffet's phenomenon). Bordet (Ifi95) extended
these observations and established that immune
bacteriolysis and hemolysis required two factors—
the heat stable antibody and a heat labile factor,
which was called alexine. This term has been
replaced by the present name compfeme/if which
was coined by Ehrlich, because this factor
complemented the action of antibody.
Bordet and Gengou [1901) described the
complement fixation Test, usir:^ the hemolytic
indicator system, as a sensitive serological reaction.
Tbi-i found wide application, and the Wasscrmann
complement fixation test for syphilis became one
of the most popular serological tests. For the next
half century, interest in complement remained
confined to its use as a tool in serological reacuons.
Since then the structural and functional complexities
of the complement system have been defined and.
its role as a mediator and arnph lier of many 1 mmunc
and inflammatory reactions recognised, The
complement system belongs to the group of
biological effector mechanisms [called tniigcmd
enzyme cascades) which also includes coagulation,
fibrinolyi ic and kirdn systems. Such biological
cascades have distinct advantages. For example, each
enzyme in the cascade is able to activate many
molecules of the succeeding component providing
for amplification of the response at each step. Every
step has its own control mechanisms so that the
cascade can be regulated with precision.
G bn raai P r o ^ e s t i £a
Complement is. present in the sera of all mammals
and also in that o f most lower animals, Including
birds, amphibians and fishes. It is a nonspecific
serological reagent in that complement from one
Species can react with an t ibodie i from other species,
though the efficiency of icacnon is influenced by
the taxonomic distance between the species.
Complement constitutes about five per cent of
normal serum proteins and is not iricreased as a
result of immunisation.
Though some of its components are heat stable,
complement as a whole is heat labile* its cytolytic
activity undergoing spontaneous denaturation
slowly at room temperature and being destroyed
in 30 minutes at 56 °C. A sc rum, deprived of
its complement activity by heating at 56 °C for
30 minutes, is then said to be 'inactivated'.
Complement (C) ordinarily does not hind to
free antigen orannbody but only to antibody which
has combined with its antigen. Various terms such
C o p y rig h te d m aterial
jis fvation, binding vt ranjcimpftfm have been used:
to re.-Jc;r to (He combination of C with LioUrtd.
Immunoglobulin, leading to foe Ktimtion <>t' the
classical C pathway, All classes of Ig do not fix C-
Only IgM, IgG 3,1 and 2 fin that order) fix C, but
not IgC4, IgA, Igl) or IgE.The site ofC binding
is located on the F t piece of [lie Ig molecule (CH '
domain on IgG. CH* on JgM), and is expressed
unly when Ig is combined with its antigen. The
fixation of C is- not influenced by the nature of
antigens, but only by foe class of immunoglobulins.
The complement system consists of at least twenty
chemically and immunologically distinct scrum
proteins comprising the complement components,
the properdin system anti the- control proteins.
Complement is a Complex of nine different
fractions called C l to C9. The fraction C l occurs
in scrum as a calcium ion dependent complex, which
on chelation with EDTA yields three protein
subunits called C lq, r, i»Tid *. Thus C is made up of
a total of 11 different piotc ins, C tractions are named
C 1 TO C9 in the sequence o f the cascading reaction,
except that C4 comes after C l, before C2.
Hie model traditionally used to explain C activity
in immune cytoJysis the lysis of erythrocyte
sensitised by its antibody. The erythrocyte (E)
antibody (A) complex is called EA, and when C
components are attached to EA, the product is
called EAC, followed by the Components that have
reacted (for example, EAC 14235 or EAC 1-5).
When a C component acquires enzymatic or other
sFetnoosrrabk- biological activity, it i:> indicated by
a bar over the component number, for example, foe
enzymatically activated C l is shown as C l .
Fragments cleaved from C components- during the
cascade are indicated by small letters (C3a, £J3b).
Inactivated forms of C components are indicated
by the prefix T*{iC3b).
Complement is normally present in the hodv in an
inactive form but when its activity is induced
by antigen-antibody combination or other stimuli,
C components react in a specific sequence as a
cascade. Basically, the C cascade is pi scries of
reactions in which the preceding components act
as enzyme:, on the succeeding components, cleaving
them into d issim ilar fragments. T h e larger
fragments usually ioin rhe cascade- The smaller
fragments which are released often possess
biological effects which contribute to defence
mechanisms hv amplifying rhe inflammatory
process, increasing vascular permeability, inducing
smooth muscle contraction, causing chemntaxis of
leucocytes, promoting virus neutralisation,
detoxifying endotoxins and effecting the release of
histamine from mast cells.
The C cascade can be triggered off by two
parallel hut independent mechanisms or pathways
which differ only in the initial steps. Once C3
activation occurs, the subsequent steps are common
in both pathways, which have been called the
classical C pathway and the alternative or properdin
pathway.
The c lass ical pathway is so called because it
was the first one identified. But actually it is a more
recently evolved mechanism of specific active
immunity, while the alternative pathway represents
a more primitive system of nonspecific innate
im m unity.
The chain of events in which C components react
m a specific sequence following activation of C l
jjid typically culminate in immune cytotysis is known
as the classical pathway (Fig. 14.1), It consists of
the following stepsi
1. The first step is the binding of C 1 to ibe
antigen-antibody complex (traditionally
represented as EA), The recognition unit of C l
is C l q . which reacts with the Fe piece o f bound
IgM or IgG, C lq has six combining sites.
Effective activation occur* only w hen C lq is
attached to immunoglobuhns by at least two of
C o p y rig h te d m aterial
 its l>i riding sires. One molecule uf IgM or iwu
molecules of [gG can therefore in inure rhe
process. C lq binding In the presence of calcium
ions le.ids to sequerLtial activation oFC li mid s.
2. Activated t ’ t s is an esterase (C I s esterase), one
molecule at winch can cleave several molecules
ofC 4 - an instance of amplification. C4 is split
into C4a, which is an anaphylatoxin, and C4b
which binds co cell membranes along with C l.
3. C 14 b in the presence of magnesium ions cleaves
C2 into C3a, which remains Iinted to cell-
bound U 4b, anti C2b ■which is released into fluid
phase. C4b2a has enzymatic activity and is
referred to as the classical pathway C3
COrivert is tf.
4r C3 convcrtase splits C3 inm? two fragments :
U3a which is an anaphylatoxin^ and C3b which
remains celt-bound along wirh C4l>2a to form
a trimokcular complex C*lb2a3b which has
enzymatic activity and is called C5 tonvcmsc,
5. The membrane arrack phase of complement
activity begins at this stage with C5 COriVertate
cleaving C 3 into C5a, an aruphytatoxin which
is released into the medium, and U5b which
continues with the cascade. C6 and C7 then
join together. A heat stable It imolecular complex
£5ti7 formed, part ot which hinds to [lie cell
membrane and prepares it for lysis by C 9 and
U9 which join the reaction subsequently- Most
of C5b7 escape and serve to simplify the
reaction by adsorbing onto Urisen si tised
'bystander cells" and rendering them susceptible
to lysis by C8 and C9, The unbound CS£ j has
chemotac t ic activity, though the effect Is
transient due to its rapid inactivation. The
mechanism of complement mediated cytolysis
is chc production of "holes', approximately 100
A in diameter on the cell membrane. This
disrupts the osmotic integrity of the membrane*
leading to the release of the cell contents.
Although the classical pathway is generally
activated by Ithe anti gen-ant iboidy Complexes or
aggregated immunoglobulin, activation may also
be due to other stimuli, such as DNA, C-reaciive
protein, trypsin-like enzymes or some retroviruses.
T h e Central process in the com plem ent cascade is
the activation ofC_\ which is the mayor component
o f C, In rhe classical pathway, activation of C3 is
achieved hy U42 (classical C 3 couvertase). The
activation ufCJ without prior participation ofC l42
is known as the ';Li(emative pathway'.
The first example o f the alternative pathway was
the demonstration bv LTI enter (1954) of the
'properdin system1 as a group of serum proteins
contributing to antimicrobial defence without
requiring spec! tic antibodies. The activator in this
system was zymosan, a polysaccharide from the
yeast cell wall, but many other substances can also
activate the pathway. These activators include
bacterial endotoxins, IgA and 1), the cobra venom
factor and the nephritic factor (a protein present in
the serum of glomerulonephritis patients).
The lirst step in tlie alternative pathway is the
binding of C3b to an activator. C3b is continuously
generated in small quantities in tiie circulation but
in the free slate it lSrapidly inactivated by the serum
protein factors 11 and 1, Bound C3b which is
protected frotn such inactivation interacts with a
serum protein tilled ]■actor B (also known as 'C3
proactivator) to form a magnesium-dependent
complex 'C 3b,dL. This, complex is cleaved iby
mother -ierurn protein Factor D (also called 'C3
proactivator coovertue*) into two fragments - Ba
and Bb. Fragment Ba is released into the medium.
Fragment Bb remains bound to C3bhforming the
esterase C3bfBb complex, which is the alternative
pathway U3 cunvcrrasc. This enzyme C3b,Bb is
cxticmcly labile. The function of properdin (also
called Factor P) is to stabilise the C3 convcrtasc,
which hydrolyses C3, leading to further steps in
the ciscade. a* in the classical pathway (Fig. 14.2).
Unchecked complement activity can cause not only
C o p y rig h te d m aterial
 ihl o imh i Mt:srr srsraw
c 1% F. *
nnmTsis
oJiiusrn.ni of the complement system hut also
serious damage to tissues. Several inbuilt control
mechanisms regulate the complement cascade it
different steps. These are mainly of two kinds:
inhibitors which hind to complement components and
halt their hirthcr fimedon, and inactivators wltieh are
enzymes that destroy complement proteins.
A . I n hibitors
1. formal serum contains an inhibitor of Cl
esterase ( C l s I N H ) . This licit labile alpha
rteurarninoglvenprotein ;lLhi> inhibit*; many other
esterases found in blood, such as plasmin,
kJiunngen and the H a rm a n factor. This dents not
prevent the normal progress of dtc complement
cascade hut checks its autocatalytic prolongation.
2. The S protein present in normal serum binds to
C567 and modulates the cytolytic action of the
membrane attack complex.
B . I nactivators
1, A scrum bctaglobulin, called factor/(formerly
known as C3h. (_’4h 1NAC, eonglutinogen
* eac T
activating factor or KAF), provides homeostatic
control o f C."S activation, particularly by the
alternative pathway.
2, Another beta globulin factor H acts in concert
with Factor I modulating C3 activation,
3, Anaphylatoxin in ictiv ito r is an alphaglobulin
that enzymatically degrades C3a, C4a and C5a
which arc anaphylatoxins released during the
C cascade.
4, C4 binding protein controls the activity of cell*
hound C4b.
Many other regulators of C activity have also
been reported,
B io l o g ic a l S ^ f b c t b o f C
Complement mediates immunological membrane
damage (cytolyais, bacteriolysis}, amplifies the
inflammatory response and participates in the
pathogenesis of certain hypersensitivity reactions,
h exhibits antiviral activity and promotes
phagocytosis and immune adherence. It also
interacts with the coagulation, fibrinolytic and
kiuinogcnic systems of blood.
C o p y rig h te d m aterial
114 a Thitlwffr d r'. " V" -v'-v v} .►
-----
■
--------- Sir B'

An important fuiiccion of C is ro facilitate the im m u n e com plex { T y p e I I I ) h person si tlvity

upfakc and Jr;, midi itn of pathgOgOU by ]>hagocytiC reactions.ThedcSTructicm o f ■■■■■■' :•■.•■■.■. I vritig
cells. This opsonic effect bast-ri on the presence incompatible transfusion and t jn
on the surface of phagocytic cells. (macrophages, se d o rm id p u rp u ra , ate ■ m ples o f T y p e 1J
monocytes, neutrophils and others) of complement reactions, C contributes to , : ic path sit of
receptors or CRs. Munv such receptors have been nep hro to xic n e p h ritis, . !i im m u n o lo g ica l
identifiedr such as CR I, 2, 3, 4 aid Clq, which kidney damage may also occur : ■ ahscncc r»f C -
stimulate phagocytosis and removal of immune C in required fo r the prodm t: -n o f im m u re
eomplexet. The CR 2 receptor on B cells also acts complex diseases Such as serum ■ -. km ami A rthur
as a receptor for the Epstein— Ran- virus (EBV), reaction. C , however, appear; to ptcVcnt immune
the causative agent nfinfectious mononucleosis, and complex disease by solubilising it . - h - ..: hotly
so has a rtlle l: i the pathogenesis <>t this condition. complexes and preventing precipitation in
The classical C pathway results in bacteriolysis vessels and tissues. Scrum C ....... ponn ■- arc
and cytoEysis, Cells vary in their susceptibility tn decreased in many ibNinnium- .ii-.i i-. -. such as
complement mediated lysis. Gram negative bacteria system ic lupus erythem atosus and rheum atoid
ire generally sensitive to lysis, while t iram positive a rth ritis , C also plays a n ■■■ -r role in the
cells are killed without lysjs. The neutralisation of pathogenesis o f autoim mune ■ lytii anemia,
viruses under some conditions requires the paroxysm al n o ctu rn a l I e in o g lo b in u rii and
participation of C, hereditary angioneurotic edema.
C fragments- released during the cascade reaction E n d o to x in is an e fficie n t acTivinoi o f the
help in amplifying the inflammatory response. C2 alternative C pathway In ■ ! ■: xic shade there is
kini ns arc vasoactive amines and increase capillary massive G 3 fixation and platelet a d h m n c t Largv
p erm eab ility. C 3 a and C 5 a . are an n p hvlato xic scale platelet Ivsis and release of Lugi: amounts n f
{histam ine releasing) and ch em o tictic. C 5 f i7 c- platelet i : ■ Lead ■ dissem irat d p itru iscular
chem oturtle and also brings about reactive lysis. coagulation ;md thm m bw ytopcnla- t iram ficjt-iri\ t
L" participates in the cytotoxic Hype II) and sepriccm iai and r| . ■ ; i i >'. iiri. syndrome

hmc t'.lh
^ muL i ■ .licit bj
i jli ,.,o it jiiuJ r

m Sl-il' 11-rf.Uu-htT1 ■ I '■■ ■■!

■' Ifli KCIVHli'r ■i ... .ii.

Oh-. B •• ----

J'wtonr tl
■Jr
B- (" —* t'lh E!h Prnrwnftn —— * |Cjtb. flh F

ci
/
I .IS. .irtr

C cp y righted m aterial
 1 C l inhibitor
11 Larly components of classical
pathway C l , C 2 , L'4
HI C 3 and its regulatory protein
C 3 b inactivator
IV C 5 to C 8
V C9
may have a similar pathogenesis. Depletion a f C
protects the SchwartEtnan reaction^
C bound To antigen-antibody complexes adheres
to crythroevKS or to nonpr unite platelets. This
reactionhC&Ued im m u n e a d h eren ce > contributes to
defence against pathogenic microorganisms since
such adherent particles arc rapidly phagocytosed.
C3 and C4 are necessary for immune adherence.
Rovi ne scira m contai ns an unusual pratei n called
congludnin (K) which causes clumping of particles
or tells coated with C , a process known as
conglutination. Congludnin reacts exclusively with
bound C3. T h o u g h conglutinin behaves as an
antibody to C, it is not an immunoglobulin and
requires C a++ for its activity. Antibodies with
conglutin in-like activity' (immunocongjutinifi, IK)
can be produced by immunisation with complement
coated materials. They may also occur frequently
in human beings and o th e r m a m m a ls as
autoantihidies to feed C The titres of SOtum IK
rise in conditions such as infections and
autoimmune diseases associated wirh increased
fixation of C in vivo. High IK levels have been
noticed in the saliva and jejunal secretions, They
are IgA antibodies whose significance is not
known.
Complement activity of scrum is measured by
estimating the highest dilution of serum lysing
Hereditary angiiineuratic edema
b LK and other collagen
vascular diseases
Severe n^urranr pyogenic
infections
Bacteremia mainly with Gram
negarive diplococci, toxoplasmosis
No particular disease
sheep erythrocytes sensitised by antieryskrucytic
.intibodv. Estimation of individual complement
com pone nts also uses be molytic activity i n a system
containing an excess of all complement components
except the one to he measured- C components can
be quantitated also by radial immunodiffusion in
agar but chi s method does nor differ nciate between
active and inactive fractions.
Complement components are synthesised in various
sites in [be body* Such as the intestinal epithelium
(C l), macrophages (C2, C4), spleen (C.5, C8) and
Ever fC3nC6n C9J. C is, to some extent, an ‘acute
phase substance' and rise in C levels (particularly
C4, C3, C5 and C6) is- observed during the acute
phase of inflammation.
Complete or partial deficiencies of all the classical
complement Components and several of the C
inhibitors have been described in humans or
animals. Sortie ate associated with severe diseases,
while in others clinical manifestations arc sporadic.
C deficiencies result in the host being unable to
efficiently eliminate the microbial antigens of
circulating immune complexes. Recurrent bacterial
and fungal infections and collagen diseases a^n occur
(Table 14.1).
Deficiency of the C l inhibitor is associated with
C o p y rig h te d m aterial
116 h TcKlbooii 01 Micros iology ►

h e re d ita ry anginne-H rocT e d t m a h a c o n d itio n
characterised hy episodic angioedema o f the
subcui.iiirdus i issues or of the mucosa of the
respiratory or alimentary tracts. It may be fatal when
the larynx and trachea are affected. T h e attack is
preci^irated by the local exhausth>n of the reduced
amount of the C l inhibitor present, leading to the
autoeatalytic activation of C l and the tinrcsrrained
breakdown o f C 4 and C2. ^'he trai:i mediator o f
the edema appear to he the t 2 l*i:un released. The
attack may be created by infusion of fresh plasma
as a source of the inhibitor. Prophylactic
administration o fc p ^ lo o am inocaprnic add [or its
analogues) is useful. They are believed to inhibit
the activation of plasma tn ay: ills, thus sparing the
small amounts of the C l inhibitor present.

Further Reeding
l.tncw^y CA -ith1 PTtpvftx iTTtmunnbi<rf<ig*\2* edn- LchIm ; Cvrrmt
Trifolin^on 5- 1993, Cnrnpkm rpt (Ip/pn*c dtaJMiHittt, C’ ijit Op--* Immvn&l, 5, 33-
DM andj Snewarr 1997. /mmumitayiT. If* edn. Edinburgh: Churchi 11 Livingstone.

C o p y rig h te d m aterial
 Structure and Functions
of the Immune System
Hie lymphorcricular system iis a complex
organis at ion of cells of diverse morphology
distributed widely in different organs and tissues of
The body l for immunity. LymphoretiaitUr
cells, consist of lymphoid and reticuloendothelial
components, with clearly demarcated functions, lire
lymphoid cells - lymphocytes and plasms cells -
arc primarily concerned wirh the specific immune
response. The phagocytic cells, forming part of the
rctiiajloendothdial system, arc primarily concerned
with the scavenged functions o f eliminating etfete
cells and foreign particles. They contribute to
nonspecific imniunicy by rtinoving mierooigaalisma
from blood and, tissues. They also play a role in
specific immunity, both in the afferent and efferent
limbs of the immune response.
The functional anatomy oftltc lymphoid system
can be appreciated only against the background of
tlLt1 ' l™ component concept1 of immunity. The
immune response to an antigen, whatever its nature,
can he of two broad types—the humoral or antibody
mediated immunity (AMI) and the cellular or cell
mediated immunity (CM1). Humoral immunity in
mediated by antibodies produced by plasma cells
and present in blood and other body fluids (hence
she name ‘.humoral1 from 'humor1 die old term lor
body fluids) ■Cellular immunity is mediated directly
by sensitised lymphocytes. Cells for each o f these
components develop through separate channels and
remain independent, though they may also interact
in some Instances (Pig. 15.1).
\he lym phoid system consists of the lymphoid
cell-, (lymphocytes and plasma cells) and lymphoid
organs. Based on the different roles they perform,
lymphoid organs can be classified into the central
(primary) and the peripheral (secondary) lymphoid
organs. The central lymphoid organs ire
lym phnepithdial structures in which the precursor
lymphocytes proliferate, develop and acquire
immunological capability.The thymus and theburea.
oJ Fabridus in birds are primary lymphoid organs,
being responsible lor the cellular and hum oral
immune respmscs, respecrivcly. The equivalent of
the avian bursa in mammals is bone marrow. After
acquiring iirnnunocompecenoe, the Lymphocytes
migrate along blood and lymph streams, accumulate
m the peripheral lymphoid organs and, following
antigenic stimulus, effect the appropriate immune
response. The spleen, lymph nodes and mucustt-
associaicd lymphoid tissue (MALT) constitute the
major peripheral lymphoid organs. Lymphoid tissue
in the gut, longs, liver and bone marrow and
lymphoid collections in the adventitious tissue of
all organs jlLho form part of rhe peripheral lymphoid
system.
] lie thymus anligr develops from rhe
epichelium ol the third and fourth pharyngeal
pouches at about rhe sisth week of ge&rarion. By
the eighth week, mesenchymal stem cells
(precursors of lymphocytes) from rhe yolk sac, fetal
liver and bone marrow reach the thymus and
differentiate into the th y m ic ly m p h o id ce lls
(thymocytes) .Iris thus the ti rsr organ in all inimil
species tn become predominantly lymphoid. In
human beings, the thymus reaches its maximal
relative size just before birth. It continues: to gmw
till about cite twelftli year. After puberty, it undergoes
C o p y rig h te d m aterial
 ll:Ji | (fl JCIfc I?l- Stl( "Hr*1W* H* ' ■'
spontaneous jtfogrcsstvc involution, Indicating dial
ii function* best In early life.
I he thymus is located behind the upper part o f
the E f t r a u T T i . Aberrant thvmic tissues are often ftrurtd
in neighbouring sires. T h e thym us has two kibes
surrounded by a fibrous capsule, Septa arising from
the capsule divide the gland iitfn lobules which
are differentiated intn an outer cortex and an lnat:r
m e d u lla. T h e cortex is crow ded W ith a ctiv e ly
pit*]iterating sm all lym p h o cy te s. T h e m edulla
co nsists m a in ly o f ep ith e lia l cells and mature
lym p h o cy te s in rhe m id d le o f w h ic h arc the
H f lg s a H i c o r p u t c le s , w h ic h arc w h o r l-lik e
aggregations o f epithelial cells.
Till the l%Os, the thymus was an organ without
any recogniseJ Junction. The for tui [Out obscr VUtWIB
bv Good (1954) of thymoma and impaired
immunity in a, patient, and by Miller (1961) of
immunodeficiency in neona Lilly rhymectomi&ed
mice, paved die way for rhe understanding of the
pivotal role of the organ in the development of cell
mediated immunity". The primary function ot the
thymus is the production of thymic Lymphocytes.
It is the major site for lymphocyte proliferation in
rhe body. However, of rhe lymphocytes produced,
H iivjlted ■
only about one per cent leave the thymus. The rest
are Jeslroyed locally.The ■: I I'll i .. pparendj
wasteful process is not known. !:< 1lie thvmuSf Ilie
lymphocytes acquire new Surface [ ■..■::n "1 hy'
antigens). Lymphocytes, >ridirianed in the thymus
are called ‘thvnius ( Tj n-.U nl lympiiocvto Or
T cells'. Unlike in the p ripl jpguns lymphocys
proliferation in the thymus Is not dependent on
antigenic stimulation. In fact, p riphetul antigedic
Stimuli do not lead tp any III r n m response in the
thymus. Antigen introduced dir I ii ■■■ i ,.■ thyrmu
tTiav lead to a local immune i -r ttet
I tic thjTnusconfers mi ii '..:i 1 '.■.nr!' 1 :
on the lymphocytes dun rig, their Kta-- .n the organ
Free hymic Lymphocytes are not ....... mper cot
In the thymus they are ' e d u t t i e d ISO d i a l the)
become capable of mounting cell medi.-" ed imi uti ■
response against appropriate antigens, The
importance of thymus in lymphocyte proliferation
and development of CM I is evident from the
lymphopenia deficient graft rtje< u<>r. ami the ■".!
called ‘runt diseasc‘ seen in neon a tally
thymcctornised mice. U cfvicot l Ml is seen
i n congem tal aplasia f the [}ivtlv. . ■ n huutao beinj^i
(DiGeorge syndrome), and in mice ('nude mice').
C o p y rig h te d m aterial
 T lymphocytes are selectively seeded into certain
site^ in the peripheral lymphatic tissues, being found
in the white pulp of the spleen, around the central
arterioles, and in the paracortical areas of lymph
nodes. These regions have been termed 'thymus
dependent1as they arc found grossly depleted after
nennan! thymectomy. While thvrrieetomy affects
CM1 primarily, it also diminishes antibody response
to many types of antigens (thymus dependent
antigens) such as sheep erythrocytes and bovine
serum albumin. Humoral response to or her antigens:
is unaffected.
This is a lvmphocpitheli.il
organ arising as a pouch from the dorsal part of
the cloaca ill binds. It becomes a Lymphoid organ
by about the day 15 of embryoration, develops full
functional ability near hatching and starts involuting
by 7-1.1 weeks of age, corresponding to the age of
puberty. The bursa is also a site of lymphocytic
proliferation and differentiation. Stem cells from
the vnlk Racr fetal liver and bone marrow enter the
bursa, proliferate and develop into immuno*
competent ‘bursal lymphocytes' or B cells (II for
bursa or bone marrow). These migrate and seed
selective areas cn the peripheral lymphoid
organs— the mantle, germinal follicLei and
peri toll icular regions of [be spleen, and the far
cortical areas and medullary cords of lymph nodes.
These are known as 'hum tlependent1 or ‘thymus
independent areas', hollowing appropriate antigenic
stimulation, U lymphocytes transform into plasma
cells and secrete antibodies.
The vital role of the bursa in humoral immunity
was discovered accidentally by Click and Chang
(1956) who found that chickens bursectomised at
batching failed to form antibodies when challenged
with a bacterial antigen. Immunocompetence is
conferred on the lymphocytes by the bursa in
stages. Competence for JgM production is
acquired early (about day 14 of embryonation)
and for IgG Late (about day 21). Birds
bujscctomiscd On IH to 20 days Synthesise IgM,
but not TgG-
In humans and other mammals, rhe bone
marrow acts as the bursa equivalent. All
lymphocytes originate in the bone marrow; While
1’ lymphocytes [levelop in the thymus, B
lymphocytes develop in the bone marrow itself. In
rhe human fetus, Ptycr's patches develop and
bmphoid cells appear in the spleen and Lymph nodes
by the 20th week of gestation. From then on the
fetus is able to produce IgM and IgD- If receives
maternal IgC, but IgA and Igti jire not present, At
birth JgM production is enhanced, but IgG level
tldls steadily, [o reach minimum levels by the 3rd
mouth. IgG production then picks up and heonmes
adequate by 2 -3 years. Full immunocompetence is
attained only alter the first decade of life.
Lymph nodes ire placed along
[be COutse <>t Ivn Lplialic vessels.They are Surrounded
bv a fibrous capsule from which trabeculae
penetrate into the nodes. The nude can be
differentiated into an oum cortex and an inner
medulla. In the cortex are accumulations of
lymphocytes (primary lymphoid follicles) within
which germinal centres (secondary follicles)
develop during antigen ic vcimulation. The follicles
contain, besides proliferating lymphocytes, dendritic
macrophages which rapture and process the
antigen. In the medulla, the lymphocytes, plasma
cells and macrophages arc arranged as elongated
branching hands (medullary cords). JJTie cortical
follicles and medullary cords contain B lymphocytes
and constitute the bursa dependent areas. Between
the conical follicles and medullary cords, there is
a broad, Ill-defined Intermediate zone (puacoriieal
area) which contains T lymphocytes and
interdigitating cells. This constitutes the thymus
dependent area (Fig, 15.2),
Lymph nodes act as a filter for Lymph, each
group of nodes draining a specific part of the body,
They phagocytose foreign materials including
microorganisms.They help in the proliferation and
C o p y rig h te d m aterial
circulation ofT and H cells. They enlarge following
local &Tidgcnic stimulation.
The spleen is the Urges l of the Lymphoid
organs, tr has a capsule from which trabeculae
descend, dividing ihe organ into seven!
interconnected compartments, The branches of the
splenic orrery travel along the trabeculae, and on
Leaving them branch again to form the central
arterioles, which are surrounded hy a sheath of
lviupliiv.il |9su4 Tins part is known as the wIleic
pulp of the spleen ami iiuv m usti rute ahciut hah to
three quarters of the organ, following antigenic
stimulation.T h e central arterioles proceed onto the
red pulp, so called bec ause of The abundance nt red
blood cells in it.
The periarterial Lymphoid collections in the
white putp o( the liplecri are called the Malpighian
corpuscles or tollicLcs. Germinal centres develop
following antigenic stimulation. Surrounding the
germiml centre is a 'mantle layer1 ot lymphocytes.
Immediately outside the periarterial Lvmphatic
sheath and separating it from the red pulp lies the
marginal wnc. The lymph uric sheath immediately
surrounding the central arteriole is the thymus
dependent area of die spleen. The perifollicular
region, germinal centre arid liranrte (aver form the
bursa dependent (thymus independent} areas (Fig.
1x3).
The spleen serves as the graveyard for effete
blood cells, tia n reserve tank and settling bed for
blood and as a systemic filler for trapping circulating
blontlluirne foreign particles. "Fhe immunological
function of the spleen is primarily directed against
bl(h>dlbnrnc antigens,
TLie mucosa Iiriing the alimentary,
respiratory, genitourinary and other him in it anti
surfaces -ire constantly exposed ro n time tolls,
antigens. These areas are endowed with a rich
collection of lymphoid cells. either specialised
aggregates like tLie Fever’s patches or scattered
isolated lymphoid follicles—collectively called the
mucosa associated lymphoid tissue (MAUD Such
lymphoid tissues in the gut, from the adenoids and
tonsils to the tollicles in the colon, are called the
gut associated lymphoid tissue (GALT) and those
in the respiratory tract, the bronchus associated
lymphoid tissue (HALT).
MALT contains lymphoid as well as phagocytic
cells, Itorh B and T cells arc present. While die
predominant immunoglobulin produced in the
mucosa is secretory IgA, other immunoglobulin
classes, IgC ,IgM and lgE are also tunned Local Iv.
There appears to he a tree traffic of antigen-
apcrific effector lymphocytes between the various
mucosal and secretory areas, so that an antigenic
exposure at one site may cause production of the
specific antibody at the other mucosal and secrtiury
si Lev ,lL ci. [’his indicates [he existence of a common
mucins?! or sip refory immure system and explains
the superiority of oral or nasal immunisation over
the parenteral route for many enteric anil respiratory
irfoctions.
Lymphocytes arc small, round
cells found in peripheral blood, lymph. Lymphoid
organs and in many other tissues. In peripheral
blood, they constitute 2 0 -4 5 per cent of rhe
Leucocyte population,, while in lymph and Lymphoid
organs they form the predominant cell type. The
human body contains about 1011 lymphocytes,
approximately 10“of theta beii ig renewed dally. Only
about one per cent of the total body lymphocytes
ait present in the blood. Ehrlich (lfi79) who
introduced a staining technique for blood cells
described lymphocytes as nonmotile end cells with
no recognisable function! Lymphocytes are now
recognised as the major cellular dem ents
responsible for inununologLad responses.
According to their size, lymphocytes can he
classified into small (5^S pm), medium (&-12 pm)
and large (12 —15 pm) lymphocytes. Small
lymphocytes are the most numerous. They consist
of a Spherical nucleus with prominent nuclear
C o p y rig h te d m aterial
chromatin and a thin rim of cytoplasm, containing
scattered ribosomes but virtually devoid of
endoplasmic reticulum or other organelles. They
arc capable of slow motility and during irrtrvrment
assume a 'hand mirror1 form, with the nucleus in
front and the cytoplasm as a tail behind.
Depending on fheir lifespan, they can be
classified as short-lived and long-lived. lyrnphEK-ytes.
In human beings, the short lived lymphocytes have
a lifespan of about two weeks, while the long lived
cells may last for three years or mote, or even ifor
life. Short lived lymphocytes are the effector cells
in immune response, while the long lived cells ai t
as the storehouse for Immunological memory Long
lived cells arc mainly thymus derived.
Lymphopoiesis takes place mainly Lu [lie central
lymphoid organs where they differentiate and
injure before entering the circulation and then the
peripheral lymphoid organs and tissues like a
policeman on heat patrol. These populations of
lym pfuKTtcs do not re Ala in distinct but mb: together
flg. 1 ( 2 MpgifiHMilk! section of tympJi nod* (an
in a process known as ‘lymphocyte recirculation'.
There is a constant traffic of lymphocytes through
the blond, lymph, lymphatic organs and tissues.
T iiis reeifctiktioii ensures that following
Lnrnoductinn of antigen into any pari o f the body,
lymphocytes cif appropriate specificity would reach
the site during their ceaseless wandering and mount
an immune response. A lymphocyte completes one
cycle of recirculation in about one or two days.
Recirculating lymphocytes can be recruited by the
lymphoid tissues vhcnever necessary. Recirculating
lymphocytes arc mainly T cells. U cells tend to he
more sessile. Chronic thoracic duct drainage will
therefore result in selective T cell depletion.
A lymphocyte that h^ been 'educated' by tbe
central lymphoid organs becomes an
'immunologically competent cell' (ICC). Mature
l and B cells, before they encounter antigens are
called naive cells. Such cells, though not actually
engaged in an immu no logical icspon&e, are
nevertheless fully qualified to undertake such a
AFFERENT
LYMPHATIC
CAP5ULF
tpabecula
G E R M IN A L
c o r t ic a l
RARACOfltlCAL
AREA
MEDUtLAHV
CORDS
EFFERENT
LYMPHATIC
C o p y rig h te d m aterial
 toSnook ol f•Wert.........’
rapansihiliiy when approprurtth' stimulated by an
antigen. 'Diey subserve the follining timed orrs—
recognition o( antigens, storage of immunological
memory;and immune response to specific antigen’:.
Lymphocytes have antigen recognition mechanisms
on their surface, enabling each cel! to recognise
only one antigen. The reaction of an
immunocompetent ccli to its specific antigen may
be induction of cither ‘tolenoct' or the immune
response. The nature of immune response depends
on whet tret the lymphocyte is a El or T cell.
Stimulated T eel Is produce certain activation
products (lymphoktnes) and induce CM1, while
Stimulated Q cells divide and transform info plasma
cells which synthesise immunoglobulins.
A number of surface antigens or markers have
been identified on lymphocytes and other leucocytes
by means of monoclonal antibodies. These
marker* reflect (he stage of ditterenriaciem and
functional properties of the cells. As they were given
different designations bv the investigators who
*vnusi 11 i :■ ^DH'vninNuii-Tui i-,imi ivf- h>s n m
15
prepared the antibodies the same marker came to
be known by different names (T 4r Leu3, and so
on), Otdev was introduced at the International
Workshops for Leucocyte Differentiation
Antigens' bv computing the tperifidticfi of different
anti-sera. When a cluster of monoclonal antibodies
was round to Ticker with a particular untigen, it was
defined as a separate marker and given a CD (cluster
of differedtiarioE]) number. Over 150 CD markers
have been identified so far. Tabic 15,1 lists a few
CD m arkers with their cell association and
previous designations. (In spite of the CD
nomenclature, some popular old names continue
to he in use, for example, T A and TB me still in use
for C l>4 (hclpcr/inducer) and CDS (suppressor/
cytotoxic] cells).
The most clcsuwt differentiation between and
B eclb is by rheir surface markers, for example, by
demonstration of CD.l un T cells and Ig an R otlls.
Many other tests help in their differentiation (Table
15.2). These include:
TnflB£DULAH
VESSEL
TF40EC.U.A
FLAP
WHI1£. PiJLP
eowusexi
QfRVlHA^ CtNT«E
Iv-AhuTLE LAVUt
ropjf
HEP ftJLP
AL AttflfflOU
A3TEPV
C o p y rig h te d m aterial
1. T cells bind to sheep erythrocytes forming
rosettes (SRBC nr E rosette) by CD2 antigen.
Q cells do not.
2. [1 cells bind to sheep eryrhrocyres coated with
antibody and complement, Forming EA C
rosettes, due to the presence of a C3 receptor
(C R 2) on the B cell surface. This receptor
(CR2) also acts as a receptor for the Epstein-
Barr virus. T cells do not possess this.
3. B cells have immunoglobulin on their surlace-
Each B cell carries about I.0': identical Ig
molecules on its surface. The first Ig class to
appear On the B rel] huiface lh monomeric IgM.
bubsequently other classes {either IgG, IgA or
IgE) may be present, along with IgD The
surface Ig on a B cell will have only a single
anrigen specificity'. It therefore serves as the
antigen recognition unit. T cells do not have
surface Ig. Instead they have T eel! receptors
{TC R } composed df two chains of polypeptides,
linked to C D 3,
4. T cells have thymus-specific antigens, which
are absent on B cells.
5. T cells undergo blast transfirimariun on treatment
with mitogens such ;ls phytohcmagglurinin (PH A)
or Concanavalin A (Con A ), while B cells
undergo similar transformation with bacterial
endotoxins, Staphylococcus aureus [Cowan I
strain} or EB virus,
6. Viewed under the scanning microscopflTT cells
are generally free of cytoplasmic surface
projections, while B cells have an extensively
filamentous surface, with numerous microvilli,
T cell precursors from the yolk sac, fetal liver and
bone marrow migrate to the thymus during the
embryonic and postnatal stages. The earliest
identifiable cells of T lineage are the CD7+ pru-T
cells, which acquire CD 2 or entering the thymus.
They synthesise CDS in the cytoplasm and become
pic-T cells. T cell receptor (TCR) synthesis also
takes place.
TC R is a heterodimer of glycoprotein chains
expressed on theT cell surface, which in association
with CD3 acts as the antigen recognition unit,
analogous to the Ig on the surface of B cells. TCR
occurs as two pairs of glycoprotein chains, either
tip or Y&. Pfe J cells ditferen ti ate into two lineages,
expressing cither op or yS T C R chains. The laigc
majority of T cells carry dp T C R . T C R chains
contain tour separately encoded regions— V or
variable, P or diversity, J or joining and C or
constant as in the case of immunoglobulins and
hence belong to the im m unoglobulin g en e
xuperihmiiy. By reassortment of these regions a very
wide repertoire of antigen specificities can be
formed on the T cell surface (Fig. 15,4).
Contact with self antigens within, the thymus
leads to the destruction of immature T cells carrying
the corresponding TCR, Thus, self tolerance or
elimination o fT cells capable of reacting with self
antigens takes place in the thymus. But cells capable
of reacting with autoantigens continue to arise
throughout life. These potentially harmful
'forbidden clones" are deleted by antigen specific
huppresorcells. Immunocomipetence against foreign
antigens is also developed in the thymus.
T cells also develop MH.C restriction so that
CDS" cells respond only to foreign antigens
presented along with H L A Class I, and C D 4‘ cells
to those presented with HLA Clans II molecules..
Immature T cells in the thymus exhibit CD7,
2, 3, 1, 4 and ST besides T C R . On functional
maturity, thev lose C D l atid differentiate into the
two major subsets CDS 4 + or C D 4 S+. Mature
CUfl A* TCR. ftfi cells are hclpeF/induccr cells,
inducing R cell differentiation, stimulating
proliferation of CDS* cytotoxic cells, producing
lymphokines and regulating certain stages of
erythropoie&is C D 4"8" T C R dji cells are
suppresiior/cyTuToxic cells, inhibiting B cull antibody
synthesis and acting as cytotoadcelector cells. Minor
subsets of CD4* cells and CDS' cells also exist.
Small numbers of C D 4 T and CD4"8“ eelfe are
also present in circulation.
C o p y rig h te d m ateri
 TtlC function of TCR 7$ cells in not well
understood, but they ait behoved 10 be immune
surveillance cells on cplthclin.! surfaces and a form
of defense against intracellular bacteria.
Sequential antigenic changes chaiaclirrbiiig 1 cell
itLaturacum enable their easy identification. Tins, has
application in defining T cell malignancies. Acute !
ceil malignancies such as. lymphoblastic leukemia and
KTnphjTKmaH involve earlv lpcelk, pro-T veIIhanil other
immature forms. Chronic T cell malignancies like
mycosis fimguides, peripheralT cell lymphomas and
HTLV'l assoc iaCeil delLilt T tell leukemias imolve
rratiut T cells, muinly C134' cells*
T cells ire broadly classified sus regulatory and
eflertur cells. Based on their surface imrfces, target
cells and functions the following T L-cll category
have been identified:
1. Hclper/induecr cell (TH ), with CD4 surface
marker, M H t clans U reJtricticm; generally
stimulating iirtl promoting the growth ofT ccfi>
.ii:d macrophages. Based mi the different profiles
of cytokines produced, two subsets are identified.
T H 1 an d T H 2.
T H l cclfe produce mainly the cytokines
interferon gamma lEf’N-y) and inttffcukin-2
(11.2) winch activate macrophages and 1 ad Is
promoting CMI, destruction of target cells and
killing of intiaccllular microbes, such as
tubercle and. lepra bacilli.
TH 2 cells produce ms inly the cytokines IL4, 5
and 6 which stimulate B cells to form antibodies,
2. Suppressor T Cell (Ttf): the^e have CI3fl surface
CD 1 Thymocytes, hangerhaw cells
CD 2 T «3| SR RC receptor
CD .1 T cell antigen receptor complex
CD 4 Helper T cell (receptor for HIV"
CD 8 Suppitmi/cytotwiic T cells
CD l^f B cells
murker :md MHC class ! reHtricti nr. They down
legulate immune responses and cheek over
stimulation.
3, Cytotoxic/cytolytic T cell (T.) with C DS surface
]Darker ,mJ MHC class 1 restriction. These can
kill and lyse target cells earning new or foreign
antigen:', inclulling tumour, allograft and virus
infected cells.
4. Memory celh "I’m Itolh CI^4 ami CDS cells
provide memory and anamnestic immune
responses.
B lymphocyte precursors, pro-li cells, develop in
the feral liver during embryonic life and in the bone
marrow afterwards continuously throughout life.
Kearrangemeni of Immunoglobulin genes takes
place cm their becoming pre-11 cells, which
synthesise cytoplasmic IgM. In the next stage -
immature H cells - [gM is expressed on the cell
MUrtace. These Cells itiigrate to the periphery and
undergo irnmunogLobului isotypesivi telling so that
instead o f IgM alone, the cell expres-Kcs on its
surface lgD, as well one of the other Ig classes—
IgM JgG , ItiAor LgE By nrassonment oflggenes,
R cells develop the capac itv to produce Ig molecu les
which can react with all the possible epitopes. By
a process of aHebe exclusion, each B cell becomes
programmed to form only one class of lg, with either
kappa and iambda light chain, possessing specificity
to a single epitope alone, and to express it on the
cell surface. By contact with self antigens during
TG, Leu ft
T il, Leu 5
T3, Leu 4
T4, Leu 3
rn$. Leu 2
R4, Leu U
C o p y rig h te d m aterial
 CD 3 receptor * - —
Surface immunoglobulins - + -

Receptor for Fc piece of IgG 4

EAC rosette
{ G receptor; CR2; T’BV lecqitnr} - 4 -

5RBC rosette (Cl>2; mcasks receptor) * - -

Thymus,-specific anrigens -I- - -
Numerous microvilli, on surface *
Blast transformation with: " "

a. anti-CD 3 * - -
h. auii-Ie - * -
e. PH A * - -

d- CootamavaJim A * - -

e- Endotoxins - 4 -

Phagocytic action - - +
Adherence to glass surface — — +

development, self tolerance is developed by clonal
deletion or anergy.
On contact with its appropriate antigen, the
mature.' R cell undergoes clonal proliferation. Some
activated B cells become long-lived memory cells
responsible for the recall phenomenon seen on
subsequent contact with the same antigen. The
majority' of activated R cells are transformed into
plasma cells.
Plasma cell is the antibody secreting cell. It is
oval, about twice the size o f a small lymphocyte,
with an eccentrically placed oval nucleus containing
large blocks o f chrom atin located peripherally
(car[wheel appearance). The cytoplasm Is large and
contains; abundant endoplasmic reticulum and a
well-developed Golgi apparatus. It is structurally
designed to be an immunoglobulin producing
factnrv. E’ lusma cells are cud cells a ill] have a short
lifespan o f two nr three days. A plasma cell makes
an antibody o f a single specificity, o f a single
immunoglobulin class and allotype, and o f a single
light chain type only. An exception is seer in the
primary antibody response, when a plasma cell
producing IgM initially, may later be switched to
]g£5 production. ’While plasma cell ih the best antibfidjr
producing cell, lymphocytes lymphoblasts and
transitional cells may slsn synthesis Ig to some
extent,
A separate lineage o f R tells* which are
predominant in fetal and early neonatal life, caress
the T cell marker CD5 on their surface and have
been named a* Bl cells. Their progenitor cells move
from the fet.il liver r<> the peritoneal cavity where
they multiply.They secrete Low affinity polyreactivc
IgM antibodies, many'of them autoanti bodies. They
,nrc responsible for the T independent 'natural1IgM
antibacterial antibodies which appear in neonates
seemingly without antigenic stimulus, CD5+ B
cells may be relevant in die causation o f auroimmune
conditions.
When circulating lymphocytes are classified by
their surface markers into T and R cells, about
5 - 1 0 pet cent of the ccUb are found to lack features
of either type. They were called liull cc/fa Because
of rbeir morphology, they are also known as farge
granular lymphocytes (L C L ), They are nearly
double the site of the small lymphocytes, with
indented nuclei and abundant cytoplasm containing

C o p y rig h te d m aterial
several a^uroph iIic granules, CAffipOted of
m itochondria, ribosomes, endoplasmic reticulum
and Golgi apparatus, LG L arc a heterogeneous
group of cells with differences in their functional
and surface marker features. The most important
member of This group is the natural killvt (NK)
cell, Others arc the antigen dependent Cytotoxic
cells (ADCC) ami the lytnphokiiu- activated kifkr
(LAK) cells, '[‘he term NK cell is sometimes used
as a common name tor all null cells.
Natural killer cells possess spontaneous
cytotoxicity towards various target cells mainly
malignant and virus infected cells. Their cytotoxicity
is nor antibody dependent or MHC restricted- NK
activity is ‘natural1 or VkJnimmune' as it lIocv nut
require sensitisation by prior antigenic contact. NK
cells therefore form pan of the innate immune set­
up. Tliey belong to a different lineage from T and
11 cedis and are therefore normally active in ‘severe
combined immunodeficiency diseases’, in which
mature T and B cells are absent. They hiveC D 16
and CD5A on rheir surface. They bind to the
glycoprotein receptors on the surface of autologous,
as well j£ illogerek target cells and release several
cytolytic factors. One of [best, perform, which
resembles co mple ment compo nent C^, causes trans­
itjembrane ports through which cytotoxic 1actors,
such as the tumour necrosis factor beta, enter the
cell and destroy it by apoptosis (programmed cell
death). NK cell activity is augmented by Interferon.
They arc considered to be important in immune
surveillance and natural defence against virus
injected and malignant mutant Cells.
A subpopulation of L G I. h ]u>ssch*c* surface
receptors for the F t part o f Ig. They are capable of
lysing or killing target cells sensitised with Igt.!
antibodies. This antibody dependent cellular
cytotoxicity is distinct from the action ot cytotoxic
T cells, which is independent of antibody. ADCC
Cells w ere formerly called killer (K) cells but are
n o w d u n f i e d w ith N K c e lls.
1ymphokinc activated ki Her ( LA K) cells a re N K,
lymphocytes treated with interleukin-2 {IL 2),
wbieh are cytotoxic lo a wide range of tumour cells
without affecting normal cells. LAK cells have
shown promise in the treatment of some tumours
such as renal cell carcinoma. 112 also acts as a
growth factor for NK cells,
Phagocytosis is phyto-
gene ri call y the oldest defence mechanism in
animals. Originating In protozoa as a combined
mechanism tor nutrition and defence, along the
dunrut of evolution, the phag[>eytK lost its trophic-
functions with the development of digestive
enzymes- In higher organisms it specialised in the
j'emoval of foreign and autochthonous particles,
Phagfjeytic culls are the mononuclear macrophages
(ot Mood ami tissues) and the polymorphonuclear
microphaget.
T h e blood macrophages (monocytes) are the
largest of the lymphoid cells found in peripheral
blood (1 2 -1 5 |um). The tissue macrophage*
(histNKytes) arc Larger (1o-2G jam). Mononuclear
macrophage cells originate in the bone marrow from
precursor cells and become monocytes in about six
days. Monocytes in circulation have an approximate
half-life of three days. They Leave the circulation
and reach various tissues to become transformed
into macrophages, with morphological and
functional feature* characteristic of the tissues, such
a* alveolar macrophages in the lungs and Kupffer
cells L]i the Liver Tissue macrophages survive for
months. MultinueWed cells and epithelioid edis
seen in granulomatous inflammatory lesions such
as tuberculosis originate from mononuclear
macrophage cell*.
The primary function ot macrophages is
phagocytosis. These Ctlll move slowly in a
ponderous and purposeful manner, their abundant
cytoplasm thrusting out re-stless pseudopodia that
glide harmlessly past normal body cells but engulf
effclt cells And. foreign particle*. They accumulate
in areas ot inflammation or tissue damage by
chcmptaais- Particles sensitised by antibodies are
ph oticytosed more readily. The phagocytosed
particle is hold inside a vacuole (phagosome), the
C o p y rig h te d m aterial
 * Structure and Function of the Immune System ►
membrane of which fuses ^ nh a lysasome, forming
a 'phagplysosnroc*, Lysosomal enzymes digest the
particle, the remnantsbeing extruded from the cells.
While phagocytosis is an effective defence againsr
most microorganisms, some {such as the bacilli of
typh< iid, brwetloti * and tuberculo-.: s) res: st digest on
and may multiply in the cells and be transported in
them to other locations.
Macrophages express many surface receptors
including la proreins* those for the Fe part oflgG*
activated complement components and various
lymphoid ncs. \ h c J i-. a protein antigen found on
mouse macrophages. A similar protein on human
macrophages has been named Afl marker. This
appear* closely related to CR3, a. cell raeptcir for
C 3 components.
Macrophages may participate in several ways
in the induction and execution of the specific
immunc response. They trap the antigen and provide
it, in optimal concentration* to the lymphocytes.
T o o high a con cen tralion o f antigen mav he
tolerogenic, and too low a concentration may not
be immunogenic. It has also been shown that with
SOrtic arttii'ciis, prior processing by in.krrophages is
an essential prerequisite for induction of antibodies.
The processing and presentation of antigen by
the macrophage to T cells require that both the
cells jHiascss surface determinants coded for by the
same major histocompatibility complex (M H C)
genes. The T cell can accept the processed antigen
only if It is presented by a macrophage carrying on
its surface the self-MHC antigens. When the
macrophage bears a different M HC antigen, i.t
cannot cooperate with the T cells. This is A’1 H C
r e s t r i c t s in.
The functional efficiency of macrophages can
be increased in many ways. They may be 'activated'
by I . m p hok m es, complement components or
interferon. Activated macrophages are not antigen-
spealic. For example* actuated macrophages from
animals infected w ith one microorganism are
cytotoxic to Tumour cells as well as to many other
microorganisms., Actuated macrophages show
127
morphological and functional changes as compared
with un&tiinukted quiescent macrophages.They'are
larger, adhere better, spread faster on glass and are
more phagocytic. They secrete a number of
biologically active substances, including hydrolytic
enzymes, b:-ndi ng protei ns (fibronect 1n, transferri n),
tumour necrosis factor fcachectin), colony
stimulating factor (C S F ) and interleukin 1
(formerly called the leukocyte activating factor).
Interleukin 1 acts as an endogenous pyrogen and
also induces synthesis of interleukin 2 by T cells.
Interleukin 2 facilitates the activation of l 1 cells.
When Ktiftiulattd by cytopliiiic antibodies and
certain lymphohmes, macrophages become "armed1.
Such armed macrophages are capable of antigen-
specific cytoroxicny, which is important in
antitumour activity and graft rejection.
Microphages are the polymorphonuclear
leucocytes of the blood— neutrophils, cotinophlls
and basophils. Neutrophils arc actively phagocytic
and form the predominant cell tvpe in acute
inflammation. The phagocytic property of
neutrophils is nonspecific* except for its
augmentation by npsonnh. They dn not appear to
have any role in specific immune protes sc s-
Eosinoplti.lic leucocytes arc found in large numbers
in aller^i.' inflammation, para--itic infections and
around anti ^n-andbody complexes. They primar ily
inhabit tissues rather than the bloodstream. 'I'hvi.r
distinctive feature is the presence of two types of
granules— the small, round, homogeneous ones and
the large owid ones. The granules eorttnin a variety
of hydrolytic enzymes which bung about extracellular
killing af large parasites. Eosinophils possess
phagocytic activity but only to a limited degree.
Basophil leucocytes are found in the blood and
tissues (mast cells). Their cytoplasm has large
numbers of prominent basophilic granules
containing hepajin, histamine, serotonin and other
hydrolytic enzymes, Degranulatioti of mast cells,
with release of pharmacologically aenve agents,
constitutes the effector mechanism in anaphylactic
and atopic allergy.
C o p y rig h te d m aterial
128 i T fis ttb o c k o r M i c r o b i o lo g y *

M l" 'H | m i IM M IIM ^ -h S-11 M

lit )N(■
MARROW

SURFACE
CD MARKERS

FKU - T

THYMUS CVTO \ PRE - T

IMMATVkL r

m ature i

,1 4
rcR - (j TC R - ^
HEL.PERylNCHJCEP CYTOTOXIC;
iUF'FKLiiSOK.

Fir?, 15.4 t c#!i m nnilon

C o p y rig h te d m aterial
 * Structure and Functions erf Uw Immune System ►
D en d ritic cells: While macrophages jire the
ni.l:iu antigen presenting cells, another type of cell
known as the dcrdriric cell also performs rhijg
functiuu. Dendritic cells are bone marrow derived
cells of a lineage diifferent from the macrophages
and T or R lyrnphocytes. I hey possess. MHC class
II antigens bur not Fc or sheep RliC receptors or
surface immunoglobulins, They have little or no
phagocytic activity. They are highly pleomorphic,
with a small central body and many long needte-
likc processes, and arc present in the peripheral
blood and in the peripheral lymphoid organs,
particularly in the germinal areas of the spleen and
lymph nudes. Dendritic cells are specially involved
in the presentation of anrigens to 1' cells dining
the primary immune response.
T h e B cell is another an Ligeti presenting cell),
particularly duri i lg the secondary i mmune response.
Langcrhans cells in the skin possess features of
macrophages and dendritic cells. They proce^ and
present antigens that reach rhe dermis.
M ^JOIl HISTOCOMPATIBILITY
C o m p l e x <M H G >
The primary function of the immune system is the
recognition and elimination of foreign cells and
antigens that enter the body. Tissues and organs
grafted from one individual to another member of
the same species {'allograll I-. J are recognised as
foreign and rejected. It was the rarlv work o f Gurer
in the 1930a on the antigens responsible for allograft
rejection in inbred mice that led to the discovery of
the major hisloCoinp.iLihiliiy complex (Ml 1 C ) .
Gofer identified two blood group antigen
systems in mice* one of which (anu^en 1) was
common to all the strains. Antigen 2 was found
only in some strains and appeared to be responsible
for allograft rciection. This was called the H-2
anligen (H for hislucocilpltthiliLV, Chapter 20}.
The histocompatibility anngens are cell surface
antigens that induce an immune response leading
to rejection o f aUognifta. ( H - 2 antigen system was
found to be the major histocompatibility antigen
129
for mice and to be coded for by a closely linked
[tiuliiaUelic cluster of genes, which was called the
major hisCocompaubLlity complex.
The development ofeongenie and recombinant
strains of mice by Snell enabled the detailed analysis
of the various loci of this complex. {The term
congenicf means animals which differ only at a
single genmc UhilisJ. Daussct pioneered Studies OH
human leucocyte antigens, which were liter found
to be the ma:or histocompatibility antigens in
human beings.
The genetic basis of immune response, which
had been suggested by many early observatl 11 ns, was
proved by Benacerraf and colleagues, who
established that the ability to respond
immunologically to an antigen was conditioned by
spec:lie genes called the immune response i. Ir)
genes. For their work on MI 3 and the genetic
control of iiiiu.ime response, Snell, Dau&set and
Bcnaccrxaf were awarded the Nobel Prize for
Medicine in IVfMj.
Early studies on M HC were carried out in mice.
However, all species of animals (including human
tnLmg&) examined subsequently were found to
possess a similar complex of genes on a segment of
one cliromosome pair, coding for three different
classes of proteins:
L Class L proteins that determine histo­
compatibility, and the acceptance or rejection
of allografts {tissues or organs from different
individuals within the same species);
2. Class II proteins that regulate the immune
response; and,
3. Class HI proteins that include some components
of the complement system and a few uthees.
'I’bc name 'histocompatibility complex' arose
because its discovery was based on transplantation
experiments, and only later were the Other two
components of the complex hfentified- The major
antigens determining hi-wcompatibility in human
beings are alloanugens, characteristically found on
the surface of leucocytes, I luman M HC antigens
are therefore synonymous with human leucocyte
C o p y rig h te d m aterial
130 i T&xlhOok of Microbiology
antigens (HLA), and the MHC complex of genes
with rhe HLA complex.
H L A complex: The HLA complex of genes is
located On the short arm of chromosome 6 {Fig.
15,5), Ti consists of three separate dusters of genes:
1) HLA class I comprising A, B and C loci;
2) Class II or rhe D region consisting ofD R , DQ_
and DP loci', and
3) Class [II or the complement region containing
gene-t for complement components C 2 and C4
of the classical pathway, as well as properdin
factor B of the alternative pathway heat shock
proteins and tumour necrosis factors a and [1.
H L A loci are multiallelic, that mh the gene
occupying the locus can be any one of several
alternative forms (alleles). As each allele determines
a distinct product (aniig£rt), the H LA by-stem is
very pleomorphic. For example, at least 24 distinct
alleles have been identified at HLA locus A and
50 at B.
H I, A cdoLk u Ih : HLA antigens arc two'chain
glycoprotein molecules anchored on the surface
membrane of cells {Fig. 15-6)-
Class I molecules consist of a heavy pep ride
cha in (alpha chain) noncovalendy 11nlccd to a much,
smaller peptide called beta 2 j microglobulin (beta
chain). The beta cham has a constant aminoacid
sequence and is coded for by a gene on chromosome
15. The alpha chain consists of three globoid
domains (alpha ], alpha l r alpha 3) which protrude
from the cell membrane and a small length of
tra ns membrane C terminus reaching into the
cytoplasm. The distal domains {alpha 1 and alpha
2) have highly vaiuble aminoacid sequences and
are folded in form acuvirv or groove between them.
Protein antigens processed by macrophages or
dendritic cells to form small peptides ait bound to
this groove for presentation to CDS T cells. The T
cell viill recognise the antigen only when presented
as a complex with the MHC class 1 molecule and
not otherwise (M H C restriction). W hen so
presented, the CDS cytotoxic killer cell destroys
the target cell [for example, a v:.ius infected cell).
*
HLA class I antigens (A, E and C) arc found
on the surface of virtually all nucleated cells. They
are [he principal antij^cnst involved in graft rejection
and cell mediated cytotyds- Class I molecules may
function as components of hormone receptors.
HLA class II antigens are more restricted in
■distribution, being found only on cells of the
immune system— macrophages, dendritic cells,
activated 1’ cells, and particularly on B cells.
Class II antigens are hecerodimers, consisting
of an alpha and a beta chuin [Fig. 15-7). Lach chid n
has two domains, the proximal domain being the
constant region and the distal the variable,The two
distal domains (alpha 1, beta 1) constitute the
antigen-binding site, for recognition by C D 4
lymphocytes, in a fash ion si mi Iar to the rccogn ifi on
of the Claw 1 antigen peptide complex by CDS T
colls. H L A class 11 molecules are primarily
responsible for the grafi-versus-host response and
the muted leucocyte reaction (MLR).
Both class 1 and II molecules are members of
the lmmunoglnhu Iin gene supetfetmiiy. The imrnune
response (/r) genes which control immunological
responses to specific antigens arc behoved to be
situated in the H L A Class II region, probably
associated vith the DR locus. It genes have been
studiLid extensively in mice and located in the I
region of mouse M HC. They code far la (1 region
■Mociaced} antigens consisting of 1A and I E
proteins. However, the relevance of It genes in
humans is not dear.
H IA class III molecules arc heterogeneous.
They include complement components linked to
the formation ofC 3 convertases, heat shock proteins
and tumour necrosis factors. Urey also display
polymorphism.
The MHC system was originally identified in
the context of transplantation, which is an artificial
event. In the natural state* besides serving as cell
surface markets that help infected cells to signal
cytotoxic and helper T cells, the enormous
polymorphism of the M H C helps maximise
protection against microbial infections. Ry increasing
C o p y rig h te d m aterial
 hi Structural and FurtCVom of ttw Immune System * 131

stkuctuhe *,n» fvmctiom»of thi=immi nt sy si -hm

CentTjmB™ Ciass II CLasa 111 Oawi

a H

CiB OF C? TNF

Fig. ISA HLA towplw loci on dwamowne

flic specificity ntJ« l f antigens, the MHC prevents
p ib o b s with related intigcfiii; i~i.■ku- up sneaking
just host Lnmaune defences by molecular mimicry.
The primary .um of the MHC may be defence
ngfltfist rnicrohes Jind not against the graft.
Ml 1C h as been implicated in a number of
i:> ■!ii . E i i [ ] i■
iri-1Us-!;ii-.il phenomena ^Ltcll j- individual
odour* body weight in mice and egg 1at Lug in
chickens.
Antisera for ] ] LA typing were
obtained principally from multiparous women as
they tend to have antibodies to the HLA antigens
of their husbands, due Co sensitisation during
pregnancy. Monoclonal antibodies to HI .A antigens
have been developed. Typing is done serologically
by microcytotoneity, which tests for complement
itiedi al ud Ivh-i>of peri fihcral blood lyn ipht ic vtc s T-Mth
a ---t.Lnci.Lni set of typing sera. Elowever, serological
taping is not p*.-sihIe lor H I.A L) R antigens, which
are detected bt the mixed Leucocyte reaction fMLR)
and i'rimed lymphocyte typing (PLT), respectively.
Genetic methods ate being used increasingly for

Fig, ISA HU duo I tnotecub Fig. 13.7 HLA d m ■ motecite

HLA-typing In] advanced centres. These employ
restriction fragment length polymorphism (RFLP)
and gene sequence specific oligonucleotide probe
Typmj?
The H LA autigensi coded for l>y the
combination of alleles at each locus oil one ftraitd
of a chromosome pair represent the hiplotype. LTic
complete Hi .A type oi an individual comprises [tie
antigens represented on both strands of the diploid
chromosome and so will consist of TWO haplotvpus
(for example, H L A -A l, -A2; -R 7 ,-B l2j-O w 3,
-Dwfii L>w4; -Dw7- -D R l; -DR7; DQjvU Q w *
-DPw4; -DPwti).
I^ue to the extreme pleomorphisitt oJ the HLA
Eyitem, delineation of the JiJLA type provides a
method of typing of individuals, that is far more
discriminating than blood grouping. HLA typulg
is used primarily for testing compatibility between
recipients and potential donors before tissue
transplantation. Tt also has application* in disputed
parernity. As the prevalence of HLA types varies
widely between different human races and ethnic
groups, HI.A tvping is used in untilrupological
studies. Imputation sCmlies of HLA polymorphism
suggest the origin of fhe human ipccics in Africa
and cmigr.ttion as different subtypes to other
continents. An association has been observed
between HI.A. types and certain diseases. Such
diseases are generally of uncertain etiology,
associated with immunological abnormalities and
Ahkus .\K iL. 994. Ce-JJuIir and
l t 1 .W 2"! edn. Phi ladelplua: Saunders.
^ ^ L U j ’. i j ' / m j i ? u m i d c £ Y .
Chapel H andM Flaeney Essen-rrafc efCIinkai mundogj, S,Led n. London: Rtm-kwell SiTidwe,
Immunobiology,'!'4 cdn. London ; Current biology.
JanewayGA and PTrivers 19%.
ftikman M and D Verpani 1997. SaifcarwJ GininJ ImmimcJctgy. EdinbuTph ChurrHllL-Livinpttnw-
Wfcir DM ind j Stewart 1997, /jnrttunvJggy, S4 nln. Edifibw^gh: QwreNJI-LL-viflgatorK-
KJciu JK and A # hX> The HLA system- Aci'rE n g) Med343;7D2
exhibit a hereditary tendency, For example, strong
association has been found between ankylosing
kjkji idyl iris Hid HI A - K2 7, rheumatoid art! irjti s .mil
H IA - 13R4, ami many auh>i mmu nc cond irfons and
H L A -DR3.
MKC fcEflTKiGTi&H
T h e importance of M HC antigens in immune
reaction is indicated by the finding that T cells
respond to processed antigens on the macrophages
and other accessory cells only when they are
presented along with the self- MHC antigen. This
is known as MHC restriction. Both class I and class
II antigens operate in thin phenomenon. Cytotoxic
V lymphocytes from immunised mice are able To
kill and h*c virus infected target cells only when
the T cells and target cells are of the nine M HC
type, so that the T cells can recognise clans I. MHC
an tigens on the target cells. J Jclper T cells can accept
antigen presented by macrophages unlv when the
macrophages bear the same class II M l 1C
molecules on the surface. For T cells participating
in delayed type hypersensitivity the antigen has to
be presented along with class II M H C
de-term inantn.
In view of the great importance of M H C
restriction in immunological control, the Nobel
Friae for Medicine for the year 1^% was awarded
to Peter I^ohcrty and Rolf Zinkcrnagd for their
seminal contributions in this area.
C o p y rig h te d m aterial
 Immune Response
The specific reactivity induced in a host by an
antigenic stimulus is known as the immune
response. In infectionhdisease it ls generally equated
with protection against invading microorganisms,
lint the immune response bus a much wider scope
and includes reactions against any antigen, living
or nonliving. tr mav lead to consequence* that are
beneficial, indifferent or injurious to the hcrat. It
also includes the state of specific ncmreactivity
(tolerance) Induced by certain Ey]>es of antigenic
stimuli. The immune response can be of two types
— the humoral (antibody mediated) and the cellular
(cell mediated) types. The two are usually
developed together, though at times one or the
other may be predominant or exclusive. They
usually act in conjunction, but may sometimes act
in opposition.
Antibody mediated immunity' (AMI) provides
primary defcntue agaiiisr most extracellular bacterial
pathogens, helps in defence against viruses that infect
through the respiratory or intestinal tract*, prevents
recurrence of vims intections and jranicipates in the
pathogenesis of immediate (types 1, 2 and 3)
hypersensitivity and certain autoimmune diseases. CcU
mediated immunity (CMI) protects against fungi,
viruses and taniltst'ive intracellular bacterial jutliogenSj
participates in the rejection of homografts. and graft-
versus-host reaction, provides immunological
survcillancic and immunity against cancer, and mediates
the jnuliogeoesis of delayed (rype 4) hypersensitivity
and certain autoimmune diseases.
The production of a nribodies consists of three steps:
1. The entry of the antigen, its distribution and fine
in the tissues and its contact wirb appropriate
imrnuiiccumpeteflt cells (rhe afferent lamb)1,
2. The processing of antigen bv cells Hod the
control of the antibody forming process (pentnif
Junctions).
3. The secretion of antibody, its distribution in
tissues- and body fluid* and the manifestation;;
of its effects (efferen t L'nih).
Antibody product ion follows a characteristic pattern
consisting oft
a- A lag phase, the immediate Stage following
antigenic stimulus during which no antibody is
detectable in circulation.
H. A log phase in which there in steady rise in the
titre of antibodies.
c. A plateau or steady state when there is an
equilibrium between antibody synthesis and
catabolism.
d The phase of decline during which the
catabolism exceeds the production and the titre
falls (Fig, 16.1).
T h e antibody response to an in it id antigenic
stimulus differs qualitatively and quantitatively from
the response to subsequent stimuli with the same
antigen. The former is called the primary response
and the latter the secondary response (Tig. 16.2) l
TTic primary response is slow', sluggish and short­
lived, with a long lag phase and low ricre of
antibodies that does not persist for lung. In contrast,
the secondary response is prompt, powerful and
prolonged, with a short or negligible lag phase and
C o p y rig h te d m aterial
a ninth higher level of antibodies that lasts for Long
periods. The antibody tanned in the primary response
is predominantly IgM and in the secondary response
[gjG. The early antibody is man specific but less avid
than the late imtibocfy The duration of the Lag phase
and the persistence ofthe antibody vary with the nature
of the antigen. With some antigens such as diphtheria
testoid, the lag phase in the primary response may he
as Jong as 2 -1 weeks, while with pneumococcal
polysaccharide, antibodies Can be detected as early as
within a fcw hours.
A single injection of an antigen helps more in
sensitising or priming the immunocompetent cells
producing the particular antibody than in the actual
elaboration of high levels of antibody. Effective
levels of antibody are usually induced only by
subsequent injection's of the antigen. It is for tins
reason that nonliving vaccines are given in multiple
doses for active immunisation. The first injection
is known is the prim ing dose and subsequent
injections as hmufer doses. With live vaccines, ;l
single dose is sufficient as multiplication of the a temporary (all in the level of circulating antibody
organism in the body provides a continuing occurs due to the combination of the antigen with
antigen LC stimulus that acts. as both the priming the antibody. This has been called the negative
and booster dose. phase. It is followed by an increase in the tiirc of
When an antigen is injected into an animal the antibody exceeding the iinitial level.
already carrying the specific antibody in circulation,
F A f ie OF I'IGBN CFj T ;i8T JIt$
The manner in which an antigen is dealt with in
the body depends on factor; such as the physical
and chemical nature of the antigen, its dose and
route of entry, and whether the antigenic stimulus
is primary or secondary. Antigens introduced
intravenously are rapidly localised in the spleen,
liver, bone marrow, kidneys and lungs. They are
broken down by the retinilooidtrthelial cells and
excreted in the urine, about 70-80 per cent being
thus eliminated within one or two days. Tn contrast,
antigens introduced subcutaneously ire mainly
localised in the draining lymph nodes, only small
amount? being found in the spleen.
Particulate antigens are removed from
circulation in two phases. The first is the

C o p y rig h te d m aterial
 i immune Response ►
noniinrrunc phase iluring which the antigen is
enguEfed by the phagocytic celk, broken down and
eliminated. With the appearance of the specific
antibody, the pha-seot immune elimination begins,
during which antigen— antibody complexes are
formed and arc rapidly phagocytosed, resuming in
an accelerated disappearance of the antigen from
circulltittn. With soluble antigens, three phases can
be recognised— equilibration, metabolism and
immune elimination. The phase of equilibration
consi-ts of diffusion of the antigen to the extravascular
spam. During the metabolic phase, the k*el of the
antigen falls due to catabolic decay. Dining the phase
of immune elimination, them is rapid elimination of
the antigen with the formadon of antigen— antibody
complexes. Such complexes can cause 1 issue damage
and may be resporiMble for 'immune complex
diseases' such as scrum skkness-
Thc speed of elimination of an andgen is related
to the speed .u which it i& metabolised. Proton
antigens arc generally eliminated within dap or
wee les, whereas polysaccharides which arc
metabolised slowly, persist for months or pars.
Pneumococcal polysaccharide, For instance, may
persist upio 20 year;' in human beings, following a
-■ingk injection.
P r o d u c t io n o f A n t ib o d ie s
Immune response to an andgen is brought about
hy three types nf cells: antigen processing celts
(APC— principally maemphages and dendrite- cells),
T cells and E cells. The fust step is the capture and
processing of the antigens by APC and their
presentation, inassociation with the appropriate MHC
molecule, to T cells. While this step is essential for
most antigens (T cell dependent antigens such as
protLiiLS and erythrocytes}, in the case of T cell
independent antigens, such as polw-iccharidcs and
other structurally simple molecules with repeating
epitopes, antibody production does not requireT cell
participation.
Only when the processed antigen i- presented
on the surface of APC, in association with M l IC
135
molecules, to the 1 cell carrying the receptor
(TCR) for the epitope is the T odJ able to rccognbc
it. In the case of C D4 (helper T /T h[) cells, the
antigen has tn he presented completed with M HC
Class I I and for C D S (cytotoxic T /T ^ .) cells with
M HC class I molecules. B cells, which possess
surface Ig and M HC class II molecules, can also
present antigens to r] cells, particularly during the
secondary' response.
The TH cell requires two signals for activation.
The first signal is a a rnibi uadon of the T cell receptor
[TCR) with the MHC class H-completed antigen.
The second signal is interleukin-l i 11,1) which is
produced by the APC. The activated T H cell forms
interleukin-2 and other cytokines required for B cell
stimulation. These include 1L4, IL5 and I Lb which
act as E cell growth factor (ROGF) and the B cell
differentiation (actor (B tD F ) that activate E cells
which halve combined with their respective antigens
to clonallv ptuli Icrate and different iate into ant ibody-
sccce ring plasma cells- A small proportion of activated
R cells, instead of bdng transformed into plasma
oelis, become long lived memory cells producing a
secondary type of response to subsequent contact with
the amigen.
Cytmoede T (CDfl/TC) cells are activated when
they contact antigens presented along with MHC
class T molecules. They also need a second signal
1L2, which is secreted by activated T h] cells. On
contact with a target cell carrying the antigen or
its surface, the activated T c cells release cytotox'ins
that destroy the target, which may be virus infected
or tumour cells. SomeTc cells also become memory
cells [Fig. 16.3).
M ond( i a i A n t ib o d ie s
A single antibody forming cell or done produces
antibodies specifically directed against a single
antigen or antigenic determinant only. However,
antibodies produced ordinarily by infection or
antigens have multiple epitopes or antigenic
determ inants, each o f which generates separate
clones of lymphocytes. This results in anristra
C o p y rig h te d m aterial
 1 - ; ;. ■ >T1

rUML NKH^PONSh

. 1 ' . >; - : — v - ^ r :~ - - 1
I. • jttjftP Q p i W M I m d y a in it on tht c<]<

. • " ,■' . ' ,. ' ' r :ar[TC . h:'.r'. :s

'. n p l IT . iwiiwwiMNWlf 1 1 liN iiitly H T
1 Hu w»(«li<Tw>iHipr(nilil w lm llr ili niilmLiiwfiri l|tir
.. , ’ V!■. 1 : , ;. ■\ xix-:'.* .
i\ ■ ■- ■ • - , ................ ■■ , .: ; : • •' '
....... 'M': 1 : ::x x x x r r
?. :? ■~-x r‘ x ‘ ' x. ' iV' ’
. r . . Jib eoFFtrsis:
•' ' " •, xj ■ ' Tvri, - - 1 c '
. xx, ) >
f ! " ' . * ■■"f .-i..XX.'GX. . J . XX'i/j . ry''')p

C o p y righted m aterial
Containing immunoglobulins of different dosses With
specialties against different epitopes o f the snuigen.
O n the otlicr hand, when a done o f lymphocytes or
plasma cells unJerries teltdiw proliferation, as Ln
multiple myeloma, andbodieft wirh a single antigenic
specificity accumulate, Such antibodies produced by
a single done and directed against a single antigenic
determinant are called m onoclonal antibodies,
MonorJona? Jjifjhurfjcs are very useful tools for
diagnostic and research technirpies.
An ingenious method for the luge scale
production of monoclonal antibodies against any
desired antigen was developed by Kr>hI<t and Milstci 1
in 1975. In recognition of the great importance of
this hyhridnnia technology, the Nohcl Prise for
Medic ine was awarded to them in I Iybridomas
arc somatic cell hybrids produced by fasing antibody
farming spleen odk with myeloma cellx.The resultant
hybrid rerains the antibody producing capacity of the
spleen cell and the ability of the myeloma cell* to
m ultiply indefinitely (R g . 16.4).
Lymphocytes from the spleen of mice
immunised with the desired antigen are fused with
mouse myeloma cells grown in culture which do
not form immunoglobulins and are deficient in the
enzyme hypnxnnthine pbosphoribosyl transferase
(H PRT). The fused Cells ire placed in basal
culture medium (H A T medium containing
hypoKanthinc, amino pterin and thymidine) which
docs HOT permit the growth of the enzyme
deficient myeloma cells. As normal lymphocytes
cannot replicate mdcfinitely, only hybrid cells
possessing properties of both the splenic
lymphocytes and myeloma cells can grow in
culture. These hybrid cells, called hybridomas, are
cloned and examined for the production of
antibodies. Clones productng antibodies ttgrunsr the
desired antigen arc selected for continuous
cultivation. Such hyhridomas can he main Mined
indefinitely in culture and will continue co form
monoclonal antibodies. They may be also injected
intraperiio]Leahy in mice and monoclonal antibodies
may be obtained by harvesting [lie ascitic fluid
produced- Hybridomits may he frozen for prolonged
Storage . The discovery o f die hybridorna technology
for the production oi unlimited quantities erf identical
monodnna] antibrniics of rhe same Ig class, possessing
uniform specificity, affinity and other properties,
created a revolution in immunology by opening up
num erous diagno stic, therapeutic and research
applications. Monoclonal antibodies against several
antigens are now available crunmencially.
Murine monoclonal antibodies, however, proved
unsuitable for human therapeutic use because they
induce cl strong anti mouse immune response.
M orcover, the Fc piece of mouse Ig could not
initiate effector defence mechanisms in human
beings. Various modifications were introduced to
improve efficiency. Cleaved Fab fragments could
be coupled to various active substances like toxins
enzymes, radionuclides or cytotoxic drugs. Mouse
monoclonal* have been humanised by genetic
manipulation to make chimeric antibodies
Consisting of murine variable regions and human
constant regions- Grafting of murine monoclonal
Cl >H loops on a human Ig framework provides a
virtually human molecule, (The antigen binding
surface of an antibody it composed of six
hyper variable loops of ami noadds. These are called
co tapirm ejj tiri fy determining regions or CDRf).
1 lunun monoclonal antibodies have subsequently
been developed. G e n e s for particular antibody
fragments have been fused M baereriophage genes.
W hole libraries o f such antibodies have beer built
using bacteriophages. I .aige quantities o f the desired
antibody can be prepared by infecting bacteria with
the appro priate bacteriophage. Such antibody
engineering holds great pmmisc for immunotherapy
F A C T O R S I kTPL'JEM iC INO A k t ib ^ y
The immune response is
under genetic control. T h e differences in im m une
response to the same antigen shown by different
individuals in a species is determined, by genetic
d iffe re n c e s . The terms ’responder' and
C o p y rig h te d m aterial
133 * Textbook or Mtcrobiotogy ►

IMMUNE REKFnNSI

‘rtw
■InlDnnLiUn*' Hklm&pna
.ant, I iV I i.i Lm HI , i M
Jnl'lKidv ■nlitiodv

Rft 16.4 : Monoctanti antibody production by hybrtdomt

C o p y rig h te d m aterial
'nonrcHpondcr arc used to describe the individual's
^apidty to respond to a particular antigen. The Ir
(immune response) genes control this property;
The embtyo is immunologLcally immature.
The Capacity to produce antibodies starts only with
the development and differentiation of lymphoid
organs. The age at which embryos acquire
immunological competence varies with different
specie?. When the potential immunocompeterit cell
comes into contact with its specific antigen during
embryonic life,, the response ]S elimination of the
cell or induction of tolerance. This is believed to
be l]il1basis lot the nunantigejiLcity oh self antigens.
During embryonic life1 the developing lymphoid
cells come into contact wirh all rhe tissue antigens
of the body released by cellular breakdown, so that
all the clones n-jf cells that hive specificity toward?
self antigens arc eliminated.
IrmnunocompeTence is not complete at birth,
but continues to develop as the infant grows. The
infant has to depend on itself for antibody production
from 3-6 months of age, byyvhich time the maternal
antibodies disappear. However, toll competence is
acquired only by about 5 -7 years tor lgG and 10-
15 years for IgA. The ontogeny of antibody response
also depends on the antigens concerned. H cell
responses to most proteins and other T celt
dependent antigens develop early while responses
to polysaccharides and other T cell independent
antigens develop only later, usually by two years.
Most IgG antibodies to polysaccharides nrc of the
]gG2 type, and lgG2 producing lit cells are the Iasi
to mature during infancy.
\lalnutiilion affects the
immune response adversely, though serum
components necessary for immunity 3re conserved
selectively tiU the nutritional deficiency becomes
marked. Protein calorie malnutrition suppresses
both humoral and cellular immune responses, the
latter more severely Deficiences of ami noacids
{tryptophan, phenyl alanine, methionine, glycine,
iso leucine) and vi ram ins (vitamin A, and U group
factor rihoflavine, pyridoxinc, pantothenic acid.
folic acid) have beer shown to cause a decrease in
antibodv synthesis.
The humoral
immune response is better following parenteral
administration ot antigen than through oral or nasal
routes-. Large particulate antigens, such us bacteria
or erythrocytes, are more effective when injected
into tissues. The route of administration may also
influence the type of antibody produced- For
production oflgA antibodies, the oral or nasal route
is most suitable. Inhalation of pollen antigen?
induces IgE synthesis, whereas rhe same antigens
given parenteraliv lead to Igt! antibodies. With
some antigens the route of administration
determines whether tolerance or antibody response
results. Injection of protein antigens into the
mesenteric vein or intrarhytn ically usually induces
tolerance. Sulibeiger {1929} and Chase (1959)
showed that guinea pigs can be rendered Specifically
tolerant if certain antigens are leal hetore a parenteral
challenge {Sulatberger-Cbase phenomenon).
Application ol simple chemicals to the slon usually
leads to cellular immune response (delayed
hypersensitivity) rather than antibody formation.
With some antigens tile site of injection seems
relevant 1lepititis B vaccine is less immunogenic
following gluteal injection than following injection
into the deltoid. This may he due to rhe paucity of
antigen presenting cells in the gluteal fat, delaying
presentation of' antigen to T and B cells.
Antibody
response is, to an extent, dose dependent. An antigen
is effective duly above a minimum critical dose.
Further increase in dose enhances the intensity of
the antibody' response. But beyond a certain level,
increase in the dose of antigen docs not improve
the antibody response but may even inhibit it and
induce tolerance. Mice injected with (J.5 pg of
pneumococcal capsular polysaccharide produce
specific antibodies, but those injected with 50 |ig of
[he antigen not only fail to form antibodies but may
not respond even to subsequent doses of the same
antigen. The massive antigenic stimulus appeals to
C o p y rig h te d m aterial
iw*mp i.lie antibody producing system and paralyse
it. This phenomenon was designated'immunological
paralyB-Is' hy Felton (1949).
The increased antibody response ro a secondary
antigenic stimulus ius already been noticed. With
repeared antigen injections, the antibody response
incoriiRes progressivclx hut after a certain stage, no
further increase occurs.
T h e term "anamnestic re actio rf wan originally
applied to the production, in response to an
antigenic stimulus, of a heterologous hut related
antihodv that the hoir hud earlier produced- For
instance, a person who had been immunised earlier
against typhoid b acilli may tO UKtim es produce
antityphoid antibodies in response to infection with
Mime other bacterium. T h is may cause confusion
in the serological diagnosis of typhoid fever but
anam nestic reaction can he differentiated from a
true secondary response as it is trance nr. The term
'anamnestic reaction has been employed by some
ro refer to the secondary response as well, so there
is some confusion in the- use of thin term.
When two or rno n antigens
art administered simultaneously, the effects may
vary. A n tib o d ie s m ay he produced against the
ditferenr antigens j usi as though they had been given
separately., ot antibody response to ore or the nttlCI
of rhe antigens may he enhanced, or rhe response
to one or more of them may he diminished
(antigenic competition). W hen two bacterial
vaccines (for example, typhoid and cholera vaccines)
lie given in a mixed form, the antibody response to
each is nor influenced by the other. When toxoids
arc given Along w jLIl Lucreri.iI vaccines (for csam ple,
triple vaccine containing diphlhcrhi and tetanus
toxoids along with Hurd pertussis vaccine) the
response to the toxoid is p o te n tiate d . W hen
diphtheria And tetanus toxoids are given together,
with one in excess, the response to the other is
inhibited. When triple antigen is given to a person
who had earlier received a primary dose of
diphtheria toxoid, the response to the tetanus and
pertussis antigens will be dimiiuslicd. Such antigenic
competition is important, from a practical point of
view, in immunisation with polyvalent antigens. For
optimal effect, the nature and relative proportions
of the different antigens in a mixture should be
carefully adjusted.
The term adjuvant refer* to any
substance that enhances the irnmunogemeitv of an
antigen. Adjuvants may confer immunogen icily on
non antigenic substances, increase the concentration
and persistence of the circulating antibody. Induce
or enhance the degree of cellular immunity and
lead to the production of'adjuvant diseases" such as
allergic disseminated encephalomyelitis. A variety
of substances exhibit adjuvant activity.
Repository adjuvants such as aluminium
hydroxide or phosphate and incorporation of
protein antigens in the water phase of a water-in-
oil emulsion (Freund's incomplete adjuvant), dcJay
the release of antigen from the sire of injection and
prolong the antigenic stimulus. Others Such as silica
particles, beryllium sulphate and endotoxins activate
macrophages. The most potent adjuvant is Freunds
complete adjuvant, which is the incomplete adjuvant
along with a suspension of killed tubercle bacilli.
Resides increasing the humoral immune response,
it induces a high degree of cellular immunity
{delayed hypersensitivity) ?s well. As It produces a
local granuloma, ir is unsuitable for human use.
The adjuvant effect of tubercle bacilli is due to a water
soluble peptide MDP (murimyl dipeptide) which
induces good antibody response without causing
granuloma. Given in mineral oil OT as liposomes, it
also stimulates cell mediated immunity. Derivatives
of MDP are being developed for human use. Gram
negative bacilli show an adjuvant effect due to their
li|vi[x>lv>AVL-b;iridefraction. Bardetella pertussis, which
has, in addition, a tymphocytosis-priomotiug fectOT
acting on hoth T and R cell*, acts as a gpod adjuvant
for diphtlterii arid tetanus toxoids in triple vaccine.
Other adjuvants commonly Luedwith human vaccines
are aluminium hydroxide or phosphate.
These inhibit
the immune response. They Ate useful in certain
C o p y rig h te d m aterial
situations Like transplarLlaEion, when it becomes
necessary to prevent graft rejection, bbramples of
immilitCSiippresiiivt agents arc X - irradiation,
radiomimetic drugs, corticosteroids, □ntirnctabolites
and other cytotoxic chemicals, and antilymphocytic
scrum.
SuhlethaL whole body irradiation suppresses
a nfibody response. When a ntigenic stimulus follows
24 hours after irradiation, antibody piraluction docs
not occur, whereas if the antigen is administered
2^'\ davs before irradiation, the antiliody response
is actuary enhanced. The primary response is more
radiosensitive than the secondary response.
Kadioimmetic drugs are agents with an action
resembling that of X ’ray?, They belong in genera]
to the class of alkylating agents (for example,
cyclophosphamide, nitrogen mustard}. In human
beings, cyclophosphamide given for three days after
the antigen, con ml deb suppresses the antibody
response. It is much less effective when given before
the antigen.
Corticosteroids cause depletion of lymphocytes
from the blood and lymphoid organs. They also
stabilise the membranes of cells and lysosomes,
inhibiting histamine release and the inflammatory
response, They suppress antibody formation in the
rat, mouse and rabbit but are much Less effective in
guinea pigs, monkeys and human beings.
Therapeutic doses have hide effect On the antibody
formation in human beings. They inhibit the
induction and manifestations o f delayed
hypersensitivity in human beings.
Ajitimcrabolkcs are substances that interfere with
the synthesis of I >NA, KhJA or both and thus inhibit
cell division and differentiation necessary' for humoral
and cellular immune responses. They include folk
acid antagonists (methotrexate), alkylating agents
(cyclophosphamide) and analogues of purine (6-
mcreaptopurinc, azath lop tine), cytosine (cytosine
anbinoside) and uracil (5-fLuorouracil)r Many
am!metabolite's find clinical application in the
prevention of graft rejection.
The drug most widely used now for
immunosuppression is cyclosporine, a cyclic
polypeptide derived front the soil fungus To/ypo-
chthum inflitum. Ir is not cytotoxic for lymphocytes
and has no -antimitotic activity It selectively inhibits
helper T cell activity. A related drug is rapamycin,
Antilymphocyrc serum (ALb) is a heterologous
antiserum raised against lymphocytes. Antibody
prepared agjiinsr thymus cells is called
untit hyrnocyte serum (ATS). The corresponding
globulin preparations ,irc called AIX j and ATG,
They were used TO prevent graft rejection. Wl]ile
all other immunosuppressive agents have
undesirable side effects, ALS is devoid of any action
other than that on Lymphocytes, ALS acts primarily
isgjiinst T lymphocytes and therefore specifically
on cell mediated immunity Humoral antibody
response to thymus independent antigens is
unaffected and may even be enhanced. ALS acts
onlv against lymphocyte-;; in circulation and not cells
m lymphoid Organs. Ah ALS is a foreign protein,
its effect is decreased on repeated administration,
which may also lead to serum sickness, and oilier
hypersensitivity reactions. Monoclonal antibodies
against specific lymphocyte membrane antigens
have been prepared.
The humoral immune
response Loan antigen can he suppressed specifically
by passive administration of the homologous
antibody. The action appears to be by a feedback
mechanism. The primary response is more
Susceptible to inhibition than the secondary
response. The antibody may also combine with the
antigen and prevent its availability for the
immunocompetent cells. The Inhibitory effoct of a
passively administered antibody OTt the humoral
1mmune response has been applied ia the prevention
of Rh sensitisation in Rh negative women catiying
Rb positive fetuses. This is achieved by the
administration of anti-Rh globulin immediately
following delivery (within 72 hours),
This effect is also relevant in the practice of
combined immunisation as in diphtheria and
tetanus. In such cases, the toxoid andi antitoxin
C o p y rig h te d m aterial
142 * Texmook o' Microbiology
should he given at separate -Iils. Adsorbed toxoid
should be used as the inhibitory cflfco is much less
than with fluid tumid-
Inrr.LU-m/.Js admnihtiatnin of immune gkdjulin. lias,
been shown to haw: imrnimnrnndulatmy effete It has
been used in the treatment of many diseases of presumed
irniriufHjpcdiolpg^ etki >gy such as thiwibocytopcni-is
and autoimmune hei i :]\tic flitemi.Lfi.
Si JPBRANT1GBNS
Superartiu^ns are certain pnutcin molecules, such
as staphylococcal enterotoidfis that activate very
large numbers of T cells irrespective of tlieir
ailtigcnic specificities. While conventional antigen
fragments bind to the a(3 hetemdimer groove of
the M H L ’ molecules through the V regions ol
T C R a and [1 chains, superamigens bnid directly
to the lateral aspect o f the T C R [1 chain. Up to 20
pci cent of the circulating T cells may be so
activated, compared to conventional antigenic
stimuli which involve only about 0-001 per cent of
them. This exaggerated J cell activation leads to
massive outpouring of T cell cytokines, causing
multisystem dysfunctions, such as seen in
staphylococcal toxic shock syndrome.
M it o g e n s
Mitogens are certain substances chat induce dh ision
of lymphocytes and other cells. Some of these, like
the lect iii glycoproteins bind to sugars on the surface
of responsive cells and activate them, causing a
ptdvclonal reaction. At low concentrations, they
stimulate B cells without polyclonal activation.
Lipopolysaccharide is such a B cell mitogen.
Some Large molecules with repealing epitopes,,
such as pneumococcal polysaccha fide directly interact
with B cell surface immunoglobulins, leading to an
IgM immune response. SuchT-independent immune
response wirh IgM antibody, is not associated either
with Ig class switching or memory.
CELLULAR IMMUNE RESPONSE
The term 'cell mediated immunity1 (CM1) refers
to the specific immune responses that do not
*
involve antibodies- hot long the only demonstrable
facet of C M I win the phenomenon of delayed,
hypersensitivity (DH) which resulted in injury
lather titan protection. The first description of a
C\11 response was the observation by Jen n er
(17V8) that inoculation of vaccinia virus :ii an
immune individual led to a local erythematous
papule in 24-72 hours. J le called this the 'reaction
of immunity'. Koch (1 8 9 0 ) described the
exaggerated cutaneous reaction of tuberculous
guinea pigs to the intradcrmal injection of tubercle
bacillus or a protein extract of the bacillus
{tuberculin}. Thereafter, the tuberculin test became
the paradigm for DH. The term 'delayed
hypersensitivity1 refers to the appearance of a skin
lesion 48-72 hours after administration of the
antigen. The lesion is an indurated nodule with
infiltration hy mononuclear cells. DH was found
to be Immunologically spec!lie but It did not have
any rclaiion to ant.bodies and could not be
transferred passively by serum- The celJular basis
ofDH was shown by Landsteiner and Chase (1942)
by its passire transfer in guinea pic- through the
injection of leucocytes from sensitised donors. With
the recognition of the two component concept of
iinmu ilily, DH and other types of CMI were found
to be mediated by T lymphocytes, A variety of
techniques are now available for the detection of
CMI, though they lack the sensitivity and precision
of antibody . l s s . i v s for humoral m i m u u L l v.
Sco pe of C M ]
CMT participates in the following immunological
functions:
1. Delayed hypersensitivity.
2. Im m u n ity in In fe ctio u s diseases caused hy
obligate and facultative intracellular parasites.
These include infections with bacteria (for
ex a m p le , tu b e rc u lo sis , leprosy, lis t e r io s is ,
brucellosis), fungi [for example, histoplasmosis,
coccidioidomycosis, blastomycosis], protozoa
(for example, leishmaniasis, trypanosomiasis)
and viruses {for example, measles, mumps).
C o p y rig h te d m aterial
 * immune R n p o 'i^ ►
3. Transplantation immunity and graft-versus-host
reaction.
4. Immunological surveillance and immunity
against cancer.
5 . Pathogenesis ofcereain autoimmune diseases (for
example, thyroiditis, encephalomyelifis).
INDUCTIOM Dl GM 1
The nature of the anrigenic stimulus is important
in the induction of CM I, It ts best developed
following infections with intracellular parasites.
Killed vaccines and other nonliving antigens do not
induce CMI unless administered with the Freund
type of adjuvants. Only T cell dependent antlgent
lead to CMI. The application of cert.Lin chein icals
on the skin induces DH.
Each T cell bears on its surface a specific
receptor (TCR) for one epitope and combines only
with antigens carrying that epitope. O n contact
with the appropriate antigen, T cells undergo blast
transform ation clonal proliferation and
differentiation into memory cetL and effector cells
providing CMI. T cells recognise antiigens only
when presented with MHC molecules. Helper T
cells react with antigens presented on the surface
of macrophages or other cells., co mplexed with
M H C class II molecules. They then release
biological mediators (lymph-oleines) which activate
macrophages, enabling them to kill intracellular
parasites. Cytotoxic T cells recognise antigen on
the surface of cells (such as virus infected, tumour
or allograft cells), in association with M CH class
1 molecules, secrete lymphokines and destroy the
target cells.
t i V T O K lM iS
Biologically active substances released by activated
T lymphocytes were called lymphokines. Similar
substances produced by monocytes or macrophages
were called monokines. Initially they were given
names based an the ir demonstrated biologi cal effects
{Table 16,1). As most lymphoid ncs exhibit multiple
biological effects and the same effect may be caused
143
Table 16-1 Examples of lymphokines
I. Affecting macrophages
a. Migrar.on Inhibiting facmr (MJF)
b. Macrophage activation/aggregation fiacKU
(M A F J
e Macrophage chemoracric factof (M C F j
II. Affecting lymphocytes
a. BlasidgKnic/mLfugtiiic factor ffJtVMh)
b T cell growth factor (TGh)
u B cell growth factor (BGF)
III. Affecting gtanulrn yr<s
a. Chemotactic factor {C F }
b. Colony stimulating factor (C S F )
IV. Affecting cultured cells
a Lymphotmdi] {LT)
b Interferon fJFN)
t T u m w i necrosis factor (T N f\l
V. Others
a. Skin reactive factor (SRJ"1
b. Transfer fector (TE)
by different lymph o k : ties, their nam es la c k
precision. The term interleukin was therefore
introduced for those products of leucocytes which
exert a regulatory in flu e n c e tin other c e lls .
Interferons, growth factors and others were found
tn have similar effects, Therefore all of them have
been grouped under the term cytokines.
Cytokines are peptide mediators or miercelluLtr
messengers which regulate immunological, inflam­
matory and reparative hoar responses. They are highly
potent hormone^like substances, active Cim at
femtomolaf (10“]JM) concenorurlitfis, They differ from
endocrine hormones in being produced ro t by
specialised glands but by widely distributed cells (such
as lymphocytes, macrophages, platelets and fibrohLEts),
and acting not systcmicaillybut locally near the producing
celt (paracrine eflkt) or directly on the produarig cells
themselves (autocrine effect). They arc, in general,
pleiotmpic, h^-ing multiple effects on the growth and
differentiation of various cell types. There is
considerable overlap in the effects produced by
different cytokines. Cloning of cytokines and the
availability of monoclonal annl>odies against them
have helped to characterise them better (Table 16.2),
C o p y rig h te d m aterial
 V INTERLEUKINS:
IL-1 (a .vul |J!■ Macrophages and other cell types Prat ferntion and differentiation ofT, B and other
cells; pyrogenic; induce acute phase protein*; bone
marrow cell firolifffiKion,
JL-.2 T celt* ProErtow growth and differentiation n f T and B
cells, cytotoxicity of T and NK. cells, secretbm ot"
other lymphokines.
1L-3 T cells M ulti C S F
1L-4 T„ ccUe Proliferation ul B and cytotoxic "I" cells; increase
Ig G I and JgE production; enhance M H C class El
and ] u;K receptor*.
ll-S T h celt* Froliferatiun ut wntKtpluIi, stimulate IgA and IgM
production.
II £ TEL macrophages, fibroblasr* Promote E5 « U differentiation; I^ G production,
acute phase proteins.
1L-7 Spleen, bone marrow stromal cells B a n d T cell nnw th faemr.
iL-e Macrophages, orhers Neutrophil chcnwttcdc factor.
IL 9 Troll T cell growth Mini prolife ritioii
1L-10 T. n cells, macrophages Irliil’it IfN production and nxmociudear cell functions.
IL-11 Bone marrow stromal cell* Induce acute phase proteins.
IL-1 J T cells Activate N K cells.
IL-1 J T cells Inhibit mononuclear n il function*.
B. CO LO NY- STIMULATING FACTORS:
g M -C S f T cells, macrophages, fibroblasts T cell and macrophage growth sriirnllatiun
□ CSF Fibroblast i. endothelium Grurukwyte growth stimulation.
M -C S F Fibroblasts, endothelium Macrophage growth atimuladon.
C. TUM OUR NECROSIS FACTORS;
T N F -k Macrophage*, ....... ocytes Lumuiir cytotoxicity, lipolysis, wasting, acute phase
proteins, phagocytic cell activation, antiviral and
antiparusitk effects, endotuxic shock.
T N F-b T cells Indue? other cytokines.
D IN T E R FER O N S
IFN a Leucocyte*
IFNb Fibroblasts Antiviral activity
IFNk T «lls Antiviral, macrophage activation; M I 1C Class 1 and
Jl expression cm cells.
E, OTHERS;
TG F b "1" and B cell* Inhibit T and B cell proliferation and hematopoiesis;
promote wound hcding.
L IF T cdls Proliferation of Sitem Cells; eosinophil chemotaxis.

The feature* of Borne important cytokines mu
presented btldW:
Originally described as the
leucucvte activating factor (LAF) in 1972 and as
the B cell activating factor {BAB) in 1974, this
cytokine was renamed interleukin-1 {IL i) In 1979.
ILl is a Stable polypeptide retaining its activity up
tej and between pH ."5-11. IL l occurs in two
molecular forms, IL l alpho and beta. I!_■1 is
principally secreted by tuticTophages and monocytes

C o p y rig h te d m aterial
but cun be produced by most ocher nucleated cells
ilso- Its production is Stimulated by antigens, toxins,
injury and inflammatory processes and in} hi ted by
cyduspuriii A, corticosteroids and pTOStagJji tldiPS.
The immunological effects of JL1 include
stimulation of T cells for the product Ion of 11.2
And other lymphokines, El cell proliferation and
ant]body synthesis, neutrophil rhemotaxis nnd
phagocytosis, Jr mediates a wide range of metabolic,
physiological, inflammatory and hematological
effects by acting on bone marrow, epithelial and
synovial cells* fibroblasts* osteoclasts, liepatotytes,
vascular endothelium and other targets- il l is an
important endogenous pyrogen. Together with the
tumour necrosis factor (TN F), it is responsible for
many of the lie macological changes in septic shock
and also enhances the initial meningeal
inflammation in bacterial meningitis. Cytokine
inhibitors Such as drsamclhasDne hare been found
to protect against the sequelae at such excessive
meningeal inflammation. On the other hand, IL l
has a beneficial effect in severe infections in
immunocompromised hosts.
The discovery in 1976 of a T
cell growth factor {T C G F) produced by Activated
T cells, which induced 1' cell proliferation and
enabled their maintenance in continuous culture,
Contributed greatly to the understanding of T tclL
functions. This cytokine, renamed 1L2* is a powerful
modulator of the immune response, tr is the major
activator nf T and B cells and stimulates cytotoxic
T cells and NK cells. It converts some mill cells
(LGL) into Lymphokiiie activated killer (LAK) cells
which car destroy NK resistant rumour cells. This
property has been used in the treatment of certain
types of cancer
1L3 is a growth factor for bone
marrow stem cells. It stimulates mult [lineage
hematopoiesis and is therefore known also as the
multicolony stimulating factor (multi-CSF).
Formerly known us the B cell
growth factor-1 (BCGF-1)* ILd activates resting
B cells and acts is a B cell differentiating factor. Ir
also acts as a growth factor for T cells and mast
cells. It enhance the action nf cytotoxic T fells-. It
may have a role in atopic hypersensitivity as ir
augments IgE synthesis.
Formerly known is the B cell
growth factor=II, JLS causes proliferation of
activated B cells. It also induces maturation of
eosinophils,
IL6 is produced by stimulated
T and B cells, macrophages and fibroblasts. It
inducts immunoglobulin synthesis by activated B
cells and formation of IL2 receptors on T cells. It
has a stimulatory effect on Itepatocytct* nerve cells
and hematopoietic cells, Tracts as an inflammatory
response mediator in host defeu ce agai 11st intactions,
These
cytokines simulate the growth and differentiation
□f pluHpotent item cells in the bone marrow. They
have been named after the types o f cell colonies
they Induce in soft agar culture— for exam ple,
granulocyte ( G ) t or m ononuclear (M ) C S F - 1 L 3
w hich htdkiCcS growth o f all types o f hem atopoietic
cells is known as m u lt i-C S F . In the body they cause
other effects also, presumably by inducing cascades
of other cytokines. They arc responsible for adjusting
the rate of production o f blood cells according to
requirements, for example! the massive granulocyte
response seen in pyogenic infections. C o lo n y
stim u li! in g factors have d in id applications for
treating hematopoietic dysfunctions in infections
and malignancies.
The
tumour necrosis factor occurs as two types, alpha and
beta. A scrum factor found to induce hemorrhagic
necrosis in Certain tumours was named the tumour
necrosis; factor.,Tlie same substance was independently
described as Cachectin, a serum fartor causing the
wasting syndrome (cachexia) during chronic
infections. This has been renamedTNFtx, Iris formed
principally hy activated macrophages and
monocytes. It resembles; IL l in possessing a very
wide spectrum of biological activities such as
participation in the manifestations of cndotoxic
C o p y rig h te d m aterial
other cytokines. T \ F p , formerly known as
lymphofnicin, it produced principally by T helper
cdl 5- Its effects are similar to those of TN Fa.
Originally identified as
antiviral agents (see chapter 49)hinterferons art now
classified as cytokines. There are three clatse?. of
IFNs, alpha produced by leucocyte*, beta produced
by fibroblasts and gamma by T cells activated by
antigenn, mitogens or exposure to I L i. IFN 7 causes
many immunological effect!;, such U macrophage
activation, augmentation of neutrophil and
monocyte function?, and antitumour acWity.
The transforming growth
factor beta (TGF|J) was so named because of its
ability to transform fibroblasts. Resides acting as a
growth factor for fibroblasts and promoting wound
healing, it also acts if a down regulator of some
immunological and hematological processes.
'Ihc leukemia inhibitors factor (LIF), produced
by T cells, helps stem cell proliferation and
eosinophil chemotuii.
Cytokine production is regulated by exogenous
stimuli such as antigens and mitogens, as well as
by endogenous factors such as neuroendocrine
hormonal peptides (corticosteroids, endorphins}
and products of lipoxygenase and cyclooxygenase
pathways. I“hey also regulate each other by posirtve
and negative feedback?. A number of cytokines (for
example, IL l, 2, 3, colony stimulating factors*
interferons} have already found therapeutic
application. With better understanding of their
properties, it is possible that many cytokines, their
agonists and antagonists could eventually be used
in the management of inflammatory, infectious,
autoimmune and neoplastic rnndirinns-
The original method for detecting CM ! was the
skin test for delayed hypersensitivity' (for example,
the tuberculin lest). A number of in vitro correlates
of CM I have now'become available, These include
the lymphocyte transformation test (transformation
ot cultured sensitised T lymphocytes on contact
with the antigen), target cell destruction (killing
of cultured cells byT lymphocytes sensitised against
them), and the migration inhibiting factor test
which is commonly employed. As originally
c ( 1i■I-l), n .:■■ . ■.>r.si-:r ,i rd incubating in .1 culture
chamber, packed peritoneal macrophage? id 1
canil ii". 11jl>t. Hit macrophages migrate to form a
bey, fan dike pattern. If the macrophages are from
a Linii - 1 ensidsed totubcncul .............iddition
o l r t ih e r c u lii I d th e 1 1? LLTf ' k . a m h e r w i f i i n h i b i t
the ■ g radon (I ig 16,5). 1’his has been d i p t r d
••«'. ’ ’ 1 f ” it" :i ;
1 C ; C ,, . | i_ I 1" . . . .. 1 1 *. . i
C o p y rig h te d m aterial
 i Immune Response ►
Ior clinical use by incubating hunun peripheral
leucocytes in capillary T u n c - in culture chambers.
When an antigen ro which the individual has CMI
is introduced into the culture medium, the leucocytes
am prevented from migrating, By comparison with
the control, it i; possible to make a semkpianEiLJtive'
UBanaanenE of the migration inhibition.
T e ia n s ^ b h F actor
Passive transfer of CMI was first achieved by the
injection of viable leucocytes from sensitised demons.
I-awrencc (1954) reported transfer of CMI in human
be mgs hy the injection of extract?: from leucocytes.
This extract is known as the "transfer factor' (TF).
The transferred iinmun i|y IS specific in that CMI
can be transferred only to those antigens to which
the donor is sensitive.
T F is a dialysable, low molecular weight
substance ( M W 2000 to 4000), res intart tn trypsin,
DNAase, R N A a s e and freeze thawing. It is stable
for several years at —20 C and in the lynphilised
firm at 4 ^C. It is iuacr irated jit 56 5C in 30 minutes.
It is not antigenic- Chemically* it appears to be a
polypeptide-polynucleotide.
T F is highly potent, an extract from 0.1 ml of
packed leucocytes being sufficient for transfer. The
transferred C M ! is systemic and not local at the
injected site alone. FollowingTF injection, DH and
various in vitro correlates of C M ! can be
demonstrated in the recipient. Humoral immunity
is not transmitted by IT, TV transfers CM! to all
the a n t igenu to whi.:h the donor is sensicive, tn
htoc. It is possible to transfer CMJ from the
recipient to another in a serial fashion.
The mechanism of action of the T F is not known.
T F could be an informational molecule or a specific
gene derepressor capable of inducing antigenieally
uncommitted lymphocytes tn produce antigen-
specitic receptors. T F activity was fiill recently
demonstrable only in human beings but it has now
been reported in monkeys, guinea pigs and mice.
T F has several applications. It has been used to
restore immune capacity m parents with f cell
147
deficiency (WLskatt-Aldri.fi syndrome). It has also
beer used in the treatment of disseminated infections
associated with deficient CMI (leprumatous leprosy,
tuberculosis, mucocutaneous candidiasis). It has been
employed 111 the treatment of malignant melanoma
and may be beneficial in other types of cancer as well.
Its use has been suggested in some autoimmune
diseases (system!■’ lupus erythematosus, rheumatoid
arthritis) and diseases of unknown etiology (sarcoidosis,
multiple sclerosis).
IMMUNOLOGICAL TOLERANCE
Immunological tolerance or immunological
uiiresponi-iveness is die condition in winch contact
with an antigen specifically abolishes the capacity to
mount an immune response agiuitst that part mi Lr
ajltijjeil when it is administered subsequently. FTns
nonreactivity is specific to the particular anLigen,
immune reactivity tn other an-igjciis being unaffected.
The first example of immunological tolerance
was the observation In- Owen 1945) o f erythrocyte
chimcrism in dizygotic catde twins, each of the
twins having erythrocytes of its own and the other’s
blood groups. As dizygotic twins are genetically
dissimilar* they do not ordinarily accept transplants
from each other but such transplants survive in
cattle twins.The reason for this tolerance was shown
to be the sharing of the same placental blood supply
by the twins during intrauterine life. Based on this
observation* Burnet and Fenner (1949) suggested
that the unresponsiveiless of individuals to self
antigens was due to the contact of the : mmature
immunological system with self antigens during
embryonic life. Any an r■L^n that comes - uro contact
with the immunological system during embryonic
life would be recognised as a self antigen and would
not induce any immune response. They postulated
that tolerance could he induced against foreign
antigens ifthey were administered during embryonic
life. This was proved experimentally by Medawar
and his colleagues (1953J using two strains of
syngeneic mice. When a skin graft from one inbred
strain of mice (CBA) iH applied or a mouse of
C o p y rig h te d m aterial
another strain (A), lt is ejected. If CBA cells arc
injected into fetal or newborn strain A mice,
however, the latter when they grow up will foeely
accept shir- grafts from CBA mice- The content of
the self antigen appears to have been enlarged hy
contact with ti foreign antigen during embryonic
life, Tltis phenomenon is called 'specific
immunological tolerance',
Developm ent of tolerance is not confine I to the
embiyo or newborn but tan occur in adults also.
Tolerance iTisv he total or partial, shor t-lived or
long-lasting. The induction, degree and duration
ol" tolerance depend on the species and
immunocompctencc of the host, nature and dose
of die antigen and the route of administration.
Rabbits and mice can be rendered tolerant more
rapidly than guinea pigs and chickens, Strain
differences in tolerance induction arc seen within
species, The higher ihe degree of immuno-
compctcncc of the host, the more difficult it is to
induce tolerance. It is fon this reason that embryos
and newborns arc particularly susceptible to
induction o f tolerance. Tolerance can he induced
in adults in whom immunneomprtence is
temporarily intermpred by immunosuppressive
agents. Induction of tolerance is very difficult in
adults already immunised against the antigen.
The physical ‘■fate of the antigen is important.
Soluble antigens and hapten v ate more tolerogenic
than partkuUtt antigens. The tolerngcmcity of an
antigen can be modified by certain procedures.
When human gaitmiaglobulin is lleJt aggregated,
it is highly immunogenic in mice hut is tolerogenic
when deaggregated. Solutions of scrum proteins
centrifuged at high speed separate into tolerogenic
supernatant and immunogenic sediment fractions.
The inductio n of td c rance is dose dependen r. "I"hr rv
is ji threshold dose, below which tolerance is not
induced. Further itlCtfca.se in dose increases the
duration of tolerance. With certain antigens,
tolerance can be induced by two types of doses, one
high and the other low, with intermediate doses
producing immunity instead of tolerance. These arc
known as 'high r.onc1 an^l low 7,onc' tolerance
rcsfjectively. A special type of high Idle tolerance
is Feltons immunological paralysis. The duration
of tolerance is variable. Tolc ranee can Ire prolonged
hy reprativl tulcrggcnic Stimuli. The route of
administration th.it best induces tolerance »s that
whereby rhe antigen wpiilihrates throughout the
extra- and intravascular compartments. W ith
Antigens that do nO| ccpi ilibra Ie readily or are rapidly
eliminated, the route of choice is intravenous.
Certain haptens [hat are immunogenic in guinea
]iigs by the intradcrmal twite art tolerogenic orally
or intravenously.
1 olerance can he overcome spontaneously or hv
in injection of cross-reacting immunogens. For
example, tolerance to bovine serum albumin in
rabbits can be abolished by i:nmunidation with
cross reacting human serum albumin. In general,
tolerance to living agents is more lasting than that
to n[inliving substances. Naturally occurring
tolerance is found in certain vital infections such
as congenital rubella and cytomegalovirus infections
m which tlwie b persistent vittiriil with a decreased
ability l or the product inn of neutralising antibodies
(penistent mlerant infection). In lymphocytic
choriomeningitis infection in carrier mice, the vims
may persist in virtually all the cells and tissues aid
be transmitted vertically to the offspring without
any demonstrable immune response or pathogenic
effect. When, [he tolerance is interrupted hy a i
induction of antibody or an injection of sensitised
Lymphocytes, disease result*.. The mechanism of
tolerance is not clear. In specific immunological
tolerance in embryonic life, the clones of cells
responding to tile particular antigen were believed
to he annihilated hy contact with the antigen. This
may not be entirely true, jih self-reactive B cells can
he (bund in adults .The more likely mechanism may
he elimination of TH cells, effectively preventing
B cell activation. This is the 'central mechanism’ of
tolerance induction. In other instances, the
mechanism may be an 'afferent block' in which
access of the antigen to immunocompetent cells is
C o p y rig h te d m aterial
int'crferod with, or an lcfforent block' in which the
antibody synthesised is neutral ised or destroyed. T
and B lymphocytes appear to possess differing
sensitivity to tokranee inductionhthe former being
mote susceptible. In general, high doses of antigen
induce B cctl tolerance and repeated minute doses
of antigen induce T cell tolerance.
Tolerance to humoral and cellular types of
immunity is usually induced simultaneously. 'Split
tolerance1 can also occur where unre-sponsiveness
is established for rme parameter of the immune
response and not to the other. In guinea pigs 1)H
to tuberculin can be inhibited, without affecting
the production of a circulating antibody, by the
injection oftubcrculoprotcin prior to immunisation
with BCG,
A succession <>f theories have been put forward
from time to time in order to explain the versatility,
specificity, memory and other features of the
Immune response. Theories of immunity' fall into
two categories: instructive and selective. The
instructive theories postulate that an
immunocompetent cell is capable of synthesising
antibodies of any specificity. The antigen encounters
an immunocompetent cell and instructs it to produce
the complementary antibody. Instructive theories
were proposed by chemists who were more
concerned with explaining the physicochemical
aspects of specificity than with the biological
principles of immune processes. Selective theories,
on the contrary, shift the emphasis from the antigen
to the immune competent cell. They postulate that
immunocompetent cells have only a restricted
immunological range. The antigen exerts only a
selective influence by stimulating the appropriate
immunn-competent cell to synthesise an antilwnly.
The first plausible theory
of immune response was the 'side chain1 theory
proposed by Ehrlich (1900). Cells were considered
to have surface 'receptors' capable of reacting with
substances having complementary'side chains’. The
physiological significance of such receptors was in
anchoring nutrients to cells before assimilation.
When foreign antigens arc intmduocd i rtfo the body,
they combine with those cell receptors which have
a complementary fit. This inactivates the receptors
and interferes with the absorption of nutrknts. As
a compensatory mechanism, there is an
overproduction of the same type of receptors, which
spill over in?to the blood and circulate as antibodies.
This was the first of the selection theories. It
explained elegantly the specificity of the antibody
response. However, when I .andsleincr demonstrated
that antibodies could be formed not only against
natural antigens but also against various synthetic
chemicals, this theory was abandoned. it was
believed that an impossibly large number of
receptors would be needed to account for the
seemingly endless scope of antibody specificity. It
ifc3 however, reinirkahle hpw closely Ehrlich
anticipated modern views on die immune response.
Instructive
theories were proposed by BreinJ and I laurowitz
(1930), Alexander (1931) and Mudd (1932),
According tn these, the antigen (or the antigenic
determinant) enters antibody forming cells and
serves as a 'template' against which antibody
molecules are synthesised so that they have
combining sites complementary to the antigenic
determinant. I’Siese are therefore known as 'direct
template1theories, Pauling (1940) presented a more
detailed model suggesting that specificity was
determined by the folding of the antibody
polypeptide chains Eo form a tertiary structure fitting
the antigenic determinant.
Burnet and
Fenner (1949) proposed this instructive theory Jfo
explain the synthesis of antibody as an adaptive
protein. They postulated that the entry of the
antigenic determinant into the antibody producing
cell induced in it a heritable change, A 'gcnocopy’
of the antigenic determinant was thus incorporated
in its genome and transmitted to the progeny cells
(indirect template). This theory explained specificity
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IK 4 IM bm k c 's'fci Y'?' e t ;

and the accordin' response Imt became untenable Burnet (1 9 5 7 )

with advances in the molecular biology ot protein proposed this theory which : iftoH immunological
ryntbesis . Burnet and Fenner were the first to explain specificity to the cellular level.
the nonjnNgcniciry (it self antigens by postulating the According Eo the clonal ;i ■ hypothesis,
embryonic recognition of 1sdf markers'. during immilnoiogioil ■■ [opmeii; cells capable
. - :■■ ■ Jerne (I 9 5 5 ) of reacting with different ‘ii were formed, by
reintroduced the concept of the selective function a process of somatic mutation. Cloi ■■ t of cells that
of antigens in his natural selection theory. T hu hod immunological reactivity ■..: i self antigens
postulated tint about a million globulin (antibody) were eliminated during unk life Such clones
molecules were formed in emhiyonic life, which are called forbidden clones- persistence or
covered the full range of antigenic specificities. development in later <tc In omstk mutation could
These globulins were the 'natural aiilib(jdLes', When lead to autoimmune processes Each
an antigen was introduced, it combined selectivelv immunocompetent cell wa ableol n'.i. gwith
with the globulin that bad the nearest one antigen (ur a small numlier ■: antigens) which
complementary 'fit'. The globulin, with the could recognise and com with antigens
combined antigen,, homed in on the antibody introduced into [he body. The .-. '-' of the contact
forming cells and Stimulated them to synthesise the with the specific antigen was -Ik i , ■ 'liter,It ■-.!
same kind of antibody. Mere, selection was to form clones synthesising the .■ t bods
postulated at the level of the antibody molecule. It The clonal selection is mure widely
did not explain the fact that immunological memory accepted than the other theories, ; iugh h i- unable
resides m the cells, and not in serum. to account for ill the features of lire immune response.

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 * Irnrrure Response ► 151

A van cty of modifications and alternative theori cs have
been pn :-|osed in recent fi mes but none has succeeded
m explaining all that is known of immunity.
As an explanation for the mechanism of
regulation of antibody r?sj»nsenJerti! has jxntulaltd
the network hypothesis. The variable region of an
immunoglobulin molecule earning the .nuLren-
combining site Is Uiffrnenl lit tliffrnent antibodieH.
'J'hc distinct ami noacid sequences at the antigen­
combining site and the adjacent parts of the variable
region are termed idiorypes. The idiotype can, in
turn , act as an antigenic determinant and induce
antiidiotypic antibodies, These in turn can induce
antibodies to them and so on, forming an idiotype
network which i- postulated to regulate the amount
of an 111'odics produced and the number of ant ibody-
forriing cells i:i action. For his theoretical
contribution to antibody formation and regulation
of the immune system, Niels KL Jeme was awarded
the Nobel Prize for Medicine in 1984.
The generic ban is of antibody diversity has been
clarified recently. An individual has the capacity to
produce an estimated 10* different antibody
molecules. To have each such .mrihodv molecule
coded for by a separate gene would require millions
of genes to be set apart for antibody production
alone. This would obviously be impossible. The
phenomenon of split genes explains this. The
genetic informal Lon for the synched is of an
immunoglobulin molecule is not present in a
conti imouR army of codonR^ Instead, this information
occurs in several discontinuous stretches of DNA
('gene segments'), each coding for separate regions
of the antibody molecule. As the constant regions
are identical lor immunoglobulins of any n n type,
there need be only one gene or a few genes for
each constant region, as ag .iinsC a very large number
of genes for the variable regions. For example, the
kappa L chain genes arc composed of three separate
segments V, J and C . There arc about a hundred
different types of V (variable) domain sequences
and only one C (constant) segment, with some five
J (joining) segments in-between. By combining
different V and J sequences with the C domain, it
is possible to provide for antibodies with at least
500 different specificities By palindromic
arrangement (sequences that can be attached by
either end), it is possible to generate many times
more different specificities. The lambda chain has
additional C sequences. The H ch.ii n gene has also
a O (diversity) segment. By the shuffling of these
different segments of the C and EE chains, it is
possible to have antil>odies with far mure than 10'
types of specificity, a total repertoire that can react
with any conceivable antigen. The split gene
shuffling takes place during cell development and
a mature R cell DNA will have only one
combination of the different segments of the
immunoglobulin gene and can therefore produce
only one type of antibody (Fig. 16.6).
Thc discovery of splir genes for
immunoglobulins demolished the long standing
dogma of Ln ne gene—<me protcii i and hn ^impumi iit
irnplicafioriR in hinlngy, beyond immunology. For
this discovery, Susumu Toncgawa was awarded the
Nobel Prize for Medicine in 19fl7.

Further Rending
VH, 1994 Hew cells pnorwE smtiRHaE, Sc^ ijt^jnc^ican 44,
JanewavCA 1993.1low the immune system recognises invaders, .^icnr Ameiican 41.
Jeme NK. 1985. The generative grammar of the immune sysrem. Sci'ener, 59,1037.
Llewellyn MB et al. 1995. Monoclonal antibodies, BMJ 505:1569,1343, 1424.
Remick EKj andJ5 Friedhnd 1997. Cytokines j'nhealth and disease, 2ndedn. ban]; Marvel-LJelJier.
Tonegawa S. 1983. Somatic generation of antibody diversity, Nature 3fl2 :575.
UA it HM .I!-..I 1 Sfcwwt 1997. Jmmn/Ki(t?gj>. 6lb «Jn. Edintnuj^i: Chiinrhi!l-Iivingihinc.
Sehwinz RS Dlversiry of the -.imimne re?ptvnw- .NVwEng J M ai ,346:1017.

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 Immunodeficiency Diseases
ImmurKKlcticicncv diseases are condition^ wKere
|hc defence mechanisms of the IwhIv arc impaired,
leading to related microbial infections of waning
severity and 'aameumes ulllnccd Hi]^L-c|iTilTiliTy to
malignancies. Deficiencies of defence mechanisms
may involve spLcilic immune function- - humoral
immunity, cell mediated immunity or both - or
nonspecific mechanisms such as phagocytutis and
complement, which augment and act in conjunction
with specific immune processes. I m mu tin
deficiencies may be classified as primary or
secondary. Primary im n u m o d cfid tn a es tv suit from
abnormalities in the development of the inimume
medlarisms. Secondary inunumKlthcicnc'tcx are
consequences of disease, drugs, nutritional
inadequacies and other processes that interfere
with the proper functioning dl the mature immune
system.
The established types of primary immurnHlrficiency
syndromes are listed in Tabic 17,1. Though primary
deficiencies o f specific immunity can be
conveniently classified as those affecting R cd l
responses, T cell responses, or both, it must be
realised that there if considerable overlapping due
to the intimate interaction between the B cell and
theT cell systems. For instance,']' ceil deficiencies
involving helper or suppressor T cclfa will have a
pro found effect on antibody response.
This
syndrome described by Bruton (1953) is the fust
lrlimuimdcLK'iLiicv disease to have been RCOglUKd
It is seen only in male infants. Manifestations are
not apparent til] about six months of age due to the
passive protection afforded by rnalem.d antibodies,
I he disease present? as recurrent serious infection*
with pyogenic bacteria, particularly with
pneumococci, streptococci, meningococci,
iVcudofntWitfl nind f / influenzae. Patients respond
normally to viral infections such as measles and
chkkcnpox, though there hjive been reports of
paralytic poliomyelitis and progressive encephalitis
following immunisation with live virus vactinre or
exposure to wild virus. As a general rule, live
microbial vaccines should not lie given, to chi hire n
with any type of primary immunodeficiency,
All classes of immunoglobulins are grossly
depleted in the serum, the IgCi level being less 1liar
a tenrh, and IgA and igM less than n hundredth of
the normal kveLTonsils and adenoids arc atrophic.
Lymph nude biopSv reveals a depletion of cells of
the bursa-dependent areais, Plasma ceils and
germinal centres are absent even after antigenic
stimulation. T h ere is a marked decrease in the
proportion of B cells in circulation. Antibody
formation dot^ not occur even after injections of
antigens.
Ceil mediated immunity' is not affected, 1 delayed
?iypt] sensitivity of tuberculin and com net dermatitis
types can be demonstrated. Allograft rejection is
normal. Arthritis, hemolytic anemia and atopic
manifestations are tn c :i|L L L ’ n t l v observed- However,
the whcal-und* flare response of atopic
hypersensitivity cannot he demonstrated.
The incidence of this condition hus been
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 4 immunodeficiency Diseases t 153

T*Ue 17.1 Clarification of primary Immunodeficiency syndromes

A. 1}r( ■:lLlis of spct Lfic immunity

1 Humoral immtmodrtidcnQ« (B cell defects)
a. X-linked agammaglobulinemia
b. Transient hypogammaglobulinemia of infancy
e, Common variable immunodeficiency nnset hypogammaglobulinemia)
d. Selective btimunogtobulin defli icndes (IgA, IgM or lgC subclasses)
er Immunodeficiencies with Hyper-lgM
£ Transect .ilirrun 11 deficiency
U. Cellular immunodeficiencies (T cell defects)
a. Thymic hypoplasia (DigeoCjje's Byrtdrortlc}
b. Chronic mucocutaneous Candida is
c. Pbrinc nucleoside phosphorylase (PNP) deficiency-
III.Combined immunodeficiencies (B and T «11 defects)
i Cellular immunodefioency with abnormal inmu noirlebuii n syntlie;ii
(M n d o f syndrome)
b. Ataxia telangiectasia
c. Wiskott-Aldrich syndrome
d Immunodeficiency with thymoma
e. Immunodeficiency with short-limbed dwarfism
£ Episodic lymphopenia with lymphocytotOKin -
g Severe comhineid immunodeficiencies
1. ‘Swiss lype’ agammaglobulinemia
2. Rcriculir dysgenesis of dc Vaal
3. AdenCMoe deaminase (ADA) deficiency
B. Disorders of complement
a. Complement component deficiencies
b. Complement inhibitor deficiencies
C- Disorders of phagocytosis
a. Chronic granulomatous disease
b Myeloperoxidase deficiency
c. Chedi-ik-Higashi syndrome
d. Leucocyte C6PD deficiency
r. Job's syndrome
i Tuftsin deficiency
g. L siy Leucocyte syndrome
h. HyperTgE syndrome
L Aetin-bindmg protein deficiency
j. Stiwachman's disease
reported as one Ln a hundred thousand population fractional catabolic rate of IgG in this condition
in the United Kingdom. Its management consists enables the maintenance of effective levels with this
of the maintenance of an adequate level of dosage. Com m e n 'L ilL preparations of
immunoglobulins. This can be achieved with an gammaglobulin oontui n only traces of IgA and JgM.
ini rial administration of3Q0 mg of gammaglobulin To provide these, whole plasma infusions have been
per kg of body weight in three doses followed by employed, the donors being tested tor hepatitis and
monthlv injections of IDO mg per kg. The slow other transmissible infections.

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Tills is due to an abnormal delay in the Initiation
of IgG synthesis in Home infants. Maternal IgG Is
sIqwIv L^tabolised in the rtewboril and reaches a
level of 200 mg per 100 ml by the second month.
Ordinarily, the infant begins synthes^mg ns own
IgG by this ager W hen there is a delay,
immunodeficiency occurs. Recurrent otitis media
and respiratory infect ions are the common disease;;
found in thin condition- Spontaneous recovery Occurs
between 18 und 30 months of age. It may be found
in infants of both sexes. Treatment with
gammaglobulin may be required in some cases but
it: is nut recommended prophylactically; as it may
contribute to prolongation of immunodeficiency by
a negative feedback inhibition of IgG synthesis.
This Common form of immunodeficiency is also
known as late onset hypogammaglobulinemia
because it usually manifests only by 15-35 years of'
age, Tt is characterised by recurrent pyogenic
infections and an increased incidence of
autoinimunti disease. Malabsorption and giardiasis
arc common. The total immunoglobulin level is
usually less tliun 300 mg j'er 100 rnh with IgG less
dun 250 mg per 100 ml. U cells may be present in
circulation in norma] numbers, but they appear
defective in their inability to ditterenfiare into plasma
cells and secrete immunoglobulins. Increased
suppressor T cell and diminished helper T cell activity
have been proposed as a cause of this disorder.
Treatment is by administration of gammaglobulin
preparations intramuscularlY or intravenously.
In these conditions, there is selective deficiency ot
one or more immunoglobulin classes, while the
others remain normal or elevated. These
‘dysg^mmaglobullnerniis’ are common and have
been reported in about one pet cent of all patients
with, recurrent infections. Isolated IgA deficiency
is the most Common condition itl this group, with
ii reported incidence nfafcKrutO.2 percent in normal
populations. These patients exhibit increased
susceptibility to respiratory infection and
steatorrhea. IgA deficiency is often accompanied
by atopic disorders. Anti-IgA antibodies arc present
in many of these patients.
Selective IgM deficiency has been found to be
associated with septicemia. Deficiencies of IgG
subclasses have been observed in relation with
chronic progressive bronchiectasis.
In
this group of immunodeficiencies, some of which
are X-l inked and some inherited as autosomal
recessive, tow IgA and IgG levels are seen with
elevated IgM. "Hu.- Ig.Vl rnolcLulej; appear to have
normal structure and possess antibody activity.
Patients show enhanced susceptibility to infections
and autoimmune processes such as
thrombocytopenia, neutropenia, hemolytic anemia
and renal lesions. Some patients develop malignant
infiltration with IgM-producing cells. Elevated
JgM level with immunodeficiency is sometimes seen
in congenital rubella.
In this
disorder, inherited as autosomal recessive, patients
show metabolic effects of mamin B12 deficiency
including megaloblastic anemia and intestinal
villous atrophy. The associated immunological
detects are depleted plasma cells, diminished
Immunoglobulin levels and impaired phagocytosis.
Treatment with vitamin Bl.2 ban been reported to
restore hematopoietic, gastnointestinal and B cell
functions but not phagocytic activity.
This is a developmental defect
involving the endtidermal derivatives of the third
and fourth pharyngeal pouches. It leads ro aplasia
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or hyjfoplasia ofdie thymus and parathyroid glands.
It docs not appear to he hereditary and does not
show a familial incidence. It in probably due to Nome
intrauterine Infection or other complications. It is
usually associated with Fallot's rerfology and other
anomalies of the heart and the jnent vessels* and a
characteristic facial ajlpeararuCc. Neonatal tetany is
present. PattenIs vvho survive the neonatal period show
enhanced susceptibility to viral, fungal and bacterial
infections, which ultimately prove fatal.
The immunodeficiency primarily involves cell
mediated immunity. The thymus dependent areas
of lymph nodes and spleen are depleted of
lymphocytes. Circulating T cells are reduced in
number Delayed hypersensitivity and graft rejection
arc depressed. The humoral immune mechanism
is largely unaffected. Antibody response to primary
antigenic stimuli is normal hut secondin' response
to many antigens is impaired. Transplantation of
fcml thymus tissue has been reported to restore the
immunological function.
This constitutes an ah norm a l im mu.no logical
response to Candida albicans. Patients develop kwcfc
chronic candidiasis of the mucosa, skin and nails.
They do not show increased susceptibility to other
infections but often have endued nopaihies. Cell
mediated immunity to Candida is deficient. In some
cases there is a total failure of T cell response to
any test antigen. Delayed hypersensitivity to Candida
antigens is absent hut circulating antibodies to them
arc found in high titres, Intracellular killing of
Candida is defective. Transfer feetOr therapy* along
with amphotericin B, has been reported effective.
The enzyme puriule
nucleoside phosphorylase is involved in the
sequential degradation erf purines to hypoxant hi ne
and finally to uric acid. Patients who have PNP
deficiency as an autosomal recessive inherited trait
show decreased cell mediated immunity acid
recurrent or chronic infections. They usually
present with hypoplastic anemia and recurrent
pneumonia, diarrhea and candidiasis. A low serum
uric acid may point to the diagnosis.
The term Nezelof
syndrome has been rather loosely applied to a group
of disorders, probably of varied etiology, where
depressed cell mediated immunity'is associated with
selectively elevated* decreased or normal levels of
immunoglobulin. The consistent features are a
marked deficiency of T cell immunity and varying
degrees- of deficiency ot B cell immunity. Indents
arc susceptible to recurrent fungal, hacterial, viral
and protozoal diseases. Abundant plasma cells arc
seen in the spleen, lymph nodes, Intestines and
elsewhere in th e body. Thymic dysplasia occurs
with lymphoid depletion. Autoimmune processes
such as hemolytic anemia are common. In spite of
normal levels of immunoglobulins* antigenic stimuli
do not induce antibody formation.
Histocompatiblc bone marrow transplantation,
transfer factor ami thymus transplantation have been
used fbr treatment, with success in some cases.
Adequate antimicrobial therapy is essential.
This is a hereditary
condition transmitted in the autosomal recessive
mode, where combined Lmmunudeficiency is
associated with cerebellar ataxia, telangiectasia,
ovarian dysgenesis and chromosomal abnormalities.
The earliest signs are ataxia and chorioathctoid
movements which are usually noticed in infancy.
Telangiectasia involving the conjunctiva, face and
other parts of the body usually appears at five or
six years of age. Death occurs due to riuoputumiiiLrv
inlettion early in life, OT malignancy in the second
or third decide. The majority of patients lack serum
and secretory IgA and some possess antibody to
IgA. IgE deficiency is also frequent, Celt mediated
immunity is also defective, resulting in an
impairment of delayed hypersensitivity and graft
rejection. Tbe disease is progressive, with both
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ne urological defects and imiouoodeficiency
becoming more severe with time. Transfer factor
therapy and f e t i d thymus t r ansplanr h have b e e n tried
with some benefil.
"J’his is an X -
linked disease characterised hy eczema,
thrombocytopenic purpura and recurrent infections.
Affected boys rarely survive the first decade of life,
dcarh being due to infection, hemorrhage or
lymphoreticular malignancy, Cell mediated
immunity undergoes progressive deterioration
associated with cellular depletion of the thymus
and the paracortioal areas of lymph nodes, fscium
IgM level is low hut IgG arid IgA levels are normal
or elevated. IsuhemaggluTinins are absent in the
serum. The humoral defect appears to he a specific
inability to respond ro polysaccharide antigens, bone
marrow transplantation and transfer factor therapy
have been found beneficial.
This
syndrome, occurring usually in adults, consists of a
benign thymic tumour, impaired cell mediated
immunity and agammaglobulinemia. 1c is frequently
accompanied by aplastic anemia. This is of historical
importance as one of the experiments of nature
winch Suggested the immunological to net ion of the
thymus.
The features of this condition arc a
distinctive form of short-limbed dwarfism,
ectodermal dvsplasta, thvmic defects and enhanced
susceptibility to intertinn. These defects arc
apparently inherited as autosomal rccessives.
In this syndrome there occurs an
episodic but profound depression o fT cell function
by the action ofa circulating compliment dependent
Ivmphocytotoxin. The toxin appears to be an
antilymphocyte antibody. The patients lack
'immunological memory' so the secondary antibody
response is abolished. The disease- is familial.
These include many syndromes with severe
deficiency of hntb humoral and cell mediated
immune responses. They arc inherited in rite
autosomal recessive mode and the primary defects
are at the level rsf the early precursors of
immunocompetent cells in [he fetal liver and bone
marrow. Many distinct patterns of severe combined
immunodeficiency have been described.
In 195 H, Swiss workers reported
agammaglobulinemia with lymphocytopenia and
severe defect in cell mediated immunity This has
been referred to as Swiij type agamma-
ghbuliim i . The basic defect is presumed to he
at the level of the lymphoid stem cell.
The most serious farm of combined
immunodeficiency is the reticular dysgenesis o f de
Viral. Here the defect is at the level of the
multipoint hemopoietic stem cell, as a result of
which there lha total failure of myelopotcsis leading
to lymphopenia, neutropenia, thrombocytopenia,
anemia and hone marrow aplasia. The condition is
[]war]ably fatal in the first week nf lifc.
Adenosme deaminase (ADA) deficiency is the
first immunodeficiency disease associated with an
enzyme deficiency. ADA catalyses the conversion
of adenosine to inosirc. art important step in the
purine metabolic pathway. How this deficiency
causes immunological impairment is not clear, f|*hc
range <*t immunodeficiency varies from complete
absence to mild abnormalities of B and T cell
functions. The condition is associated with
chondrocyte abnormalities which can be discerned
radiological!)'.
Genetic deficiencies h&ve been detected for almost
all the complement components in human beings.
The defects are transmitted as autosomal recessive
Traits. Hemolytic and other functional activities arc
completely restored by supplying the deficient
factor. CJompiement component deficiencies have
been frequently associated with systemic lupus
erythematosus. Recurrent pyogenic infections were
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found associated with C3 deficiency and nei&seriaJ
infections with deficiency o fC b , Q l and CS-
I leredirary angioneurotic edema is due to a genetic
deficiency of C l inhibitor. This relatively common
defect is transmitted as an autosomal dominant.
Androgens, aminocapmic acid and its analogue
tranexam ic aoid have been found useful in the
management of this condition. Plasma infusionh,
once recommended for treatment, have been given
up as they were found to worsen the condition in
some eases.
The rare deficiency o f C 3b inactivator has been
associated with chronic recurrent pyogenic lesions.
Phagocytosis may he impaired by either intrinsic or
extrinsic defects. Intrinsic disorders may he due to
defects witllin [he phagocytic Cell, -such as enzyme
deficiencies. Extrinsic disorders may he due to a
deficiency' of opsonic antibody, complement or other
factors promoting phagocytosis, or to the effects of
drugs or antinuutrophil autoantihodics. Phagocytic
dysfunction leads to increased susceptibility ro
infection, ranging from mild recurrent skin infections
to overwhelming systemic infection.
This
familial disease manifests itself as rcemvent infection
with low grade pthcgjens, starting early in life. The
progress is chronic and the outcome fatal, Chronic
suppurative granulomatous Eesions develop in rite skin
anil lymph notles, along with bepatosplenomeg^ly,
progressive infiltration of lungs and granulomatous
septic osteomyelitis. Humoral and cellular immune
responses are normal.
The bacteria involved in the recurrent infections
arc catalase positive pyogenic pathogens such as
staphylococci and coliforms. Catalase negative
pathogens such as streptococci and pneumocncci
are handled normally. Leucocytes from the patients
arc unable to kill catalase positive bacteria following
phagocytosis.The bacteria multiply in the cells and,
being protected from antihodies- and antibiotics by
their intracellular position, set up chronic
suppurative infection. The diminished bactericidal
capacity' of the phagocytic cells is associated with
a decrease of some metabolic processes like oxygen
■consumption* hexose monophosphate pathway
activity and production of hydrogen peroxide. The
diminished H^.Q^ production appears to be the
major reason for the bactericidal defect, The
leucocytes do not undergo degnmulation following
phagocytosis, The delayed granule rupture and
defective release of myeloperoxidase also contribute
to inefficient bactericidal activity. LeucOCyttS from
the patients fail to reduce nitroblue tetrazolium
(NHT) during phagocytosis.This property has been
used as a screening method {N B T test) for the
diagnosis of chronic granulomatous discasc.
Thc disease shows two types of inheritance-—
the more common X-linked type seen in boys and
the rare autosomal recessive type seen in girls.
In this rare
disease, leucocytes have reduced myeloperoxidase,
Patients are particularly liable to Candida albicans
infecrion.
This is a
genetic disorder characterised by decreased
pigmentation o f t h e s k i p , ey es a n d hair,
photophobia, nystagmus and giant peroxidase
positive inclusions in the cytoplasm ofkucocyies.
The inclusions may be the result of autophagocytic
activity. The leucocytes possess diminished
phagocytic activity. Patients suffer from frequent
and severe pyogenic infections.
In this rare
disease, leucocytes arc deficient in glucose 6
phosphate dehydrogenase and show diminished
b actericid al activ ity after phagocytosis. T h e
condition resembles chronic granulomatous disease
in reduced rrvycloperoxidase activity and .
susceptibility to microbial agents, but the N R T test
may be normal.
This is ch aracteristd by
multiple large ‘cold1 staphylococcal abscesses
containing large quantities of pus, occurring
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repeatedly on the nkin ;md pn various* organ;*, With
litrlc inflammatory response- Atopic eczem a,.
chronic nasal discharge and otitis media are
common features. The scrum l mmu noglobu Iins are
normal, except for elevated I g E /J ’hc pathogenesis
of the syndrome is not clear but it is probably a
primary defect in phagocytic function,
A Leucokinin capable ot
Stimulating phagocytosis, discovered at Tufts
University, Boston, has* been designated 'tuftsin*.
Chemically, it is a email teirapeptlde (Thr-Lys-
Pro-Arg), Patients with tuftsin deficiency have
been reported to be prone to local and systemic
bacterial infections.
The basic defect
here is in chemnlasis and neutrophil mobility. The
bone marrow has a normal number of neutrophils
but there is a peripheral neutropenia, with poor
leucocyte response to cliemical and inflammatory
stimulation. Patients* show an increased
susceptibility to bacterial infection, with recurrent
stomatitis, gingivitis and otitis.
These patients^ of both
sexes, have an catty onset of eczema and recurrent
bacterial infections such as abscesses, pneumonia and
secondary infection tit ecxema. The organisms
responsible include Staphylococcus a circus and
S httptpcotxvapyogan « , Cellular and humoral immune
mechanisms are normal but serum IgjE levels are
usually more than ten times the normal level.
Frequent itifecrinn and slow mobility of leucocytes
result from the defective actio-hi ndiog protein in
these patients.
In this condition,
Rosen K£ ei A 1994. The primary LmimmodefLCLcncie;- .Veu' Engl J Afed 311:235,3tXJ.
Fischer A and A Amaii-VLIlena 1995. Immunodeficiencies of genetic oripin. Immunol Today 16:510.
in n - 1 DP and Al Terr 1991. itasn' ami Clinical Immunology. 7 ' cdn. Connecticut: Appleton- Lange.
\Yizgel 11. l99i. Immunodeficiency. Q jjt Opin Immunol 5:567.
frequent infections are found together with decreased
neutrophil m o b ility, pancreatic malfunction and
bone abnormalities.
A variety of factors such as malnutrition, malignancy;
infection;*, metabolicdisorder* and cytutcncic drugs may
lead to deficits in specific and nonspecific immunity.
AIDS is a secondary immunodeficiency; Secondary
imiLiui 11 aleJ LL'icuc ics are therefore very much more
common than primary deficiencies.
Deficiencies of humoral and cellular immune
response may occur secondarily during the course
of many disease processes. Humoral deficiency
results when li cells are depleted a& in lymphoid
malignancy particularly in cEirOnic lymphatic
leukemia; when immunoglobulin catabolism is
increased as in the n e p h ro tic syndrome, when
excessive loss of scrum protein o ccu rs as in
extol i at Lve shin disease and in protein-losing
enteropathies; and when excessive production of
abnormal immunoglobulins occurs as in multiple
myeloma. Cell mediated immunity is depressed in
lympborcticular malignancies, as in Hodgkin's
disease; obstruction to lymph circulation or
lymph orrheas; when the thymus dependent areas
of lymph nodes am infiltrated with nonlymphoid
cells as in lepromatoua leprosy; and, transiently,
following certain viral infections such as measles.
Nutritional deprivation affects both types of
immune responses adversely; Ageing also causes
waning in the efficiency of acquired immunity.
Immunodeficiency follows the intentional or
unintentional administration of immunosuppressive
agents.
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 Hypersensitivity
Immunity was originally considered a protective
process, helping the body ro overcome infectious
agents and ilurir toxins. This however is only one
aspect of the broad phenomenon of immu nity wh ich
includes all manner of specific responses to antigens.
Immune response may sometimes be injurious to
the host. Sensitised individuals respond to
subsequent intigeoic stimuli in an inappropriate or
exaggerated manner, leading to tissue damage,
disease or ever death. The term hypeniensitis'ity
refers to the injurious consequences in the sensitised
host, following contact with specific antigens. In
the protective processes of immunity', the focus of
attention is the antigen and what happens ro it—
lor example, killing of a bacterium or neutralisation
of a toxin, In hypersensitivity, on the other hand,
antigens are of little concern and often, they are
innocuous or bland substance? such as serum
proteins or pollen, Elyperscnsitivicy is concerned
with what happens ro the host as a remit of the
immune reaction.
Considerable confusion is attached to the use
of rhe term 'allergy'1. As originally used by von
Pirquct, allergy meant an altered state of reactivity
to an antigen, and included both types of Immune
responses, protective as well as injurious. It is still
used in this broad sense by some. Others use the
term ‘allergy'1to mean all immune processes harmful
to the host, such as hypersensitivity and
autoimmunity. Allergy is probably most commonly
used as a synonym for hypersensitivity. It is
sometimes employed in a narrow sense to refer to
only one type of hypersensitivity, namely ‘atopy’.
For induction of hypci5cn5.itivily reactions, the host
should have had contact with the antigen, (allergen).
The Initial contact sensitises the immune system,
leading to ihe priming of rhe appropriate B or T
lymphocytes. I ll is is known as the ‘sen? irising' or
'priming1 dose. Subsequent contact with the allergen
causes manifestations of hypersensi riviry, This is
Icrujwn as the 'shocking' dose.
Hypersensitivity reactions have been classified
traditionally into 'immediate' and "delayed’ Types,
based on the rime required for a sensitised host ro
develop clinical reactions on rc-exposure to the
antigen. The major differences between the
immediate and deLaved types of hypersensitivity
reactions arc shown in Table 1,8vl
The immediate and delayed reactions are
subdivided into several distinct clinical types:
(B cell or
antibody mediated)
Anaphylaxis
Atopy
Antibody mediated cell damage
A rth u s p h e n o m e n o n
Scrum sidene^
(T cell
mediated)
Infection (tuberculin) rype
Contact dermatitis Type
Coombs and Cell (1 9 6 3 ) classified
hypersensitivity reactions into four types based on
the different mechanisms of pathogenesis. Their
classification, now widely used, is outlined below:
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1. Appears and recedes rapidly.
2. Induced by antigens or haptens by any route.
.1. Circulating antibodies present and responsible
lor traction* 'antibody mediated' reaction.
4, Pwoiv* transfer possible with KtUITI-
5. Derensitisition easy, but short-lived.
{Anaphylactic, IgE or re agin
dependent): Antibodies ('cytotropic' IgE
antibodies) arc fixed on the surface of tissue cells
(mast cells and basophils) In sensitised
individual*. The antigen combines with, the cell-
fixed antibodyT leading to release of
pharmacologically active substances (vasoactive
amines) which produce the clinical reaction,
(Cytotoxic or cell stimulating): This
type of reaction is initiated bv lgG (or rarely
lgM ) antibodies that react either with cel!
surface or ti-KHue antigen^- Cell or tissue damage
occurs in the presence of complement or
mononuclear cells* Type II reactions are
intermediate between hypersensitivity and
autoimmunity. Comhination with antibody may,
in some instances, cause stimulation instead of
damage. An example is the 'long acting thyroid
stimulator1 (LATS), an antibody against some
determinant on thyroid cells, which stimulates
excessive secretion of thyroid hormone* (Such
antibody mediated cell stimulation has also been
called type V hypersensitivity.)
(Immune complex or toxic complex
disease): Here the damage is caused by antigen
antibody complexes. These may precipitate in
and around small blood vessels, causing damage
to cells secondarily, or on membranes, interfering
with their function.
(Delayed or cell mediated
1. Appears slowly, lasts longer.
2. Antigen nr hapten intradrrmally or with
Freunds adjuvant or by skin contact
3. Circulating antibodies may be absent and
nut responsible tor reaction; 'cell mediated'
reaction.
4. Cannnot be transferred with serum; but
possible with T cdls or transfer (actor.
5. Difficult, but long-luting.
hypersensitivity): This is a. Lett mediated
response. The antigen activates specifically
sensitised CD 4 and CDS T cells, leading to the
secretion of lymphokincs, with fluid and
phagocyte accumulation.
The classification and some of the features of
hypersensitivity reactions are shown in Table
1B.2. The four types of i mmu no pathogenic
merKmifftij ilrreclbrJ are nor m utually
exclusive. Any given hypersensitive reaction may
comprise the components of more than one, or
all n f these mechanisms. The pathology and
clinical features of such immunological diseases
would also be influenced by the contributions
o f many nomni mune bodv mechanisms such as
inflammation, complement, coagulation,
fibrinolytic and kinitiogcnie systems, collectively
called the humoral amptifiattion systems.
These occur in rwo forms - the acutehpotentially
fatal, systemic form called anaphylaxis and the
chronic or recurrent, nonfctal, typically localised
form called atopy.
This is the classical immediate hypersensitivity
reaction. The term anaphylaxis (ana = without,
phyhxis - protection) was coined by Richer 0 M 2 )
to describe his observation that dogs which had
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 Type 1: IgE type 1. Anaphylaxis
2. Atopy
Type 11: Cytolytic Antibody mediated damage-
and cytotoxic TbmmboL^TOpcnia-agramuio'
iTytosls, hemolytic anemia, ere.
Type 11]: Immune 1. Arthur reaction
complex 2. Serum sickness
Tvpe IV: Delayed l . Tuberculin
hypersensitivity 2. Contact dermatitis
survived a sublcthal injection of a toxic extract of
sea anemones went; rendered highly susceptible Co
minute doses of the toxin given days or weeks latei,
instead of becoming immune tn it, Theobald Smith
(1902) had noticed a similar phenomenon in guinea
pigs, following widely spaced injections of train—
antitoxin mixtures. Ehrlich named ibis the
Theobald Smith phenomenon' and showed that it
was independent of the toxin and itltiIOXi.fi used,
since the phenomenon could be induced with
normal serum also.
Sensitisation is most effective when the antigen
it introduced parentcralLy but may occur by any
mute, including ingestion or inhalation. In
susceptible species, very minute doses Can sensitise
the host. Antigens 3S well as haptens can induce
anaphylaxis. There should be an interval of at least
2-3 weeks between the sensitising dose and the
shocking dose. Once sensitised, [he individual
remains so £br lung periods- Die shocking dose u
most effective when injected intravenously, less
effective intraperituneuLly or subcutaneously and
least effective intradcrmnlly. The shocking antigen
must be identical or immunologic ally closely
related to the sensitising antigen. The clinical
features of anaphylaxis are the same With anv antigen
bur vary between species. The clinical effects are
due to smooth muscle contraction and increased
vasci 11ar permeability. The organs affected vary with
Minutes IgR: histamine and
other pharmacological
agents
Variable: hours IgG; IgM, C
to days
Variable: hours Ig£;JgM ,C ,
to days leucocytes
Hours Co days T cvllm lyTnphukiiie&i.
macrophages
the species. Tissues or organs predominantly
involved in die anaphylactic re action are known as
L[argei tissues' or 'shock organs’. Other changes seen
in anaphylaxis are edema, decreased coagulability
of blood, fall in blood pressure and temperature,
leucopenis and thrombocytopenia.
There is considerable species variation in
susccphbil ity to anaphylaxis- Gu inea pigs are highly
susceptible and rats very resistant. Rabbits, dogs
and human beings are of intermediate susceptibility.
Anaphylaxis can be readily induced in guinea pigs.
I f a small dose of egg albumin is injected
intraperitonea 11y, followed 2-3 weeks later by a
slightly larger dose of the same antigen
intravenously the guinea pig will exhibit a dramatic
sequence of events:. Wilkin n11 miles the animal
becomes irritable, sneezeh, coughs, experiences
respiratory distress, develops convulsions and dies.
The heart continues to beat for some time after the
respiration has stopped. At autopsy, the lungs air
markedly emphysematous and do Hot collapse when
the thorax is opened or even when they are cut
into pieces.The shock o : gun is tli£ lung. Death lS
due to the constriction of the smooth muscles of
the bronchioles causing respiratory standstill.
I d rabbity death in anaphylactic shock is due
to constriction of the pulmonary artery and its
branches, leading to extreme dlLatation o f the right
side of the heart. Respiratory movements continue
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162 4 Textbook oi Microbiology >
after the cessanon of the heartbeat. In dogs, the
reaction is slower and takes 1-2 hours. There is
constriction of the hepatic venous system with gross
engorgement oftlie liver and profound tad of blood
pressure. In human beings, fatal anaphylaxis is
fortunately rare. Symptoms and signs of
anaphylactic shock begin with itching of the scalp
and tongue, flushing of the skin over the whole
body and difficulty in breatlung due to bronchial
spasm. There may he ’ i.LLLse.i, vomiting, abdominal
pain and diarrhea, sometimes with blood in the
Stool. Acute hvpolL'usiou, loss dfiionScinusness and
death follow. Human anaphylaxis once commonly
associated with heterologous scrum therapy is now
seen mostly following injections of antibiotics or
other drugs. Insect stings can also cause anaphylaxis
m human bei:igs. Prompt treatment with adrenaline
tan be life-saving. Adrenaline is to be administered,
0.5 ml of a 1 in 1000 solution, subcutaneously or
intramuscularly, the dose bring repeated upto a total
of 2 ml aver 15 mmutes, it necessary.
C u ta n e o u s a n a p h y la x is ; W hen a small
shocking dose of an antigen is administered
intradermally to a sensitised host, there will be a
local whcal-and-fUie response (local anaphylaxis).
The wheat is a pale, central area of puffiness due
to edema, which is surrounded by a flare caused by
hyperenu.i. and subsequent erythema. Cutaneous
anaphylaxis (skin test for Type I hypersensitivity)
is useful in testing for hypersensitivity and in
identifying the allergen responsible in atopic
diseases. In highly sensitised individuals, even
the skin test may lead to serious and even fatal
react Lons. Hence a syringe loaded with adrenaline
should always be kept ready whenever a skin
test is performed to detecr anaphylactic
hypersensitivity.
Efiisaive cu tan eou s, a n a p h y la x is ( i 3tl\ > :
This test developed by Ovaiy (1952) is an extremely
sensitive in vivo method for detection of antibodies.
A small volume of the antibody is injected
mcradermaJly into a normal animal. If the antigen,
along with a dye such as Evans blue, is injected
intravenously 4 -2 4 hours afterwards, there will be
an immediate blueing at the site of iutradermaJ
injection due to vasodilatation and increased
capillary permeability (wheal-and-flare reaction).
PCA can be used to detect human IgfJ antibody
which is hetcrocytotiopic (capable of fixing to cells
of other species) but not IgE which is
homocytotropic (capable of fixing to cells of
homologous species only).
A n ap h y laxis in vitro! Isolated tissues, sueh
as Intestinal or uterine muscle strips from sensitised
guinea pigs, held in a bath of Ringers solution will
contract vigorously on addition of the specific
antigen to the bath. This is known as the Schultz—
Dale phenomenon. The react ion is spec itic and will
be elicited only by the antigen to which the animal
is sensitive. Tissues from normal animals can he
passively sensitised by treatment with serum from
sensitised animals.
M ee h a n L^m of a n a p h y la x is : The
immunolo^ic basis for hypersensitivity is cytotropic
IgE antibody. Free IgE antibody in circulation is
not relevant in anaphylaxis Thus, an animal
with a h.Lgh tn:re of circulating antibody may be
refractory to shock, while anaphylaxis may be
caused by cell fixed antibody, even in the absence
of detectable circulating antibody. While in human
beings, IgE is the cytophilic antibody, in the guinea
pig and mouse the analogous cytuphilic antibody
is IgGl.
IgE molecules arc bound to surface receptor*
on mast cells and basophils. These cells cany large
numbers of such receptors called Fc ER receptors,
analogous to TC R receptions on T cell surface. IgE
molecules attach to these receptors by their Fc end.
Following exposure to the shocking dose, the
antigen molecules combine with the cell bound
IgE, bridging the gap between adjacent antibody
molecules. This tru ss- tin king increases the
permeability of the cells to calcium ions and leads
to degntnukdon, with release of biologically active
substances contained in the granules. The
manifest at ions o f anaphylaxis are due to
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pharmacological mediators, 'which arc of rwo kinds
— primary mediators which are the preformed,
eon rents of mast cell and ha so phi I granules
(hittiminti serotonin, eosinophil chemoractic factor
of anaphylaxis, neutrophil chcmotacric factor*
heparin arid various proteolytic enzymes) and
secondary mediators winch are newly formed LLpcm
stimulation by mast cells, basophils and other
leucocyte* {slow reacting suhstance of anaphylaxis,
prostaglandins .ird plateEct activating factor, and
cytokines such as 1L3, lL4t 1L5, lL6j GM-CbF).
Histamine: This is the most important vasoactive
amine in human anaphylaxis. Histamine is formed
by the decarboxvlation of histidine found in the
granules of mast cells, basophils and in platelets.
Released into ibe skin, histamine stimulates sensory
nerves, producing bunting and itching sensations.
It causes vasodilatation and hyperemia hv an axon
reflex (flare effect) and edema by increasing capillary
permeability (wheal effect). Histamine induces
smooth muscle contraction in diverse tissues and
organs, including vascularure, intestines, uterus and
especially the bronchioles. It also stimulates
HccretioTiH (secretogogue effect).
1. S e ro to n in fS -liy d ro x y try p ta n iin e ):
This is a base derived by decarboxylation of
tryptophan, [t is found in the intestinal mucosa,
brain tissue and platelets. It causes smooth
muscle contraction, increased capillary
permeability and vasoconstriction, it is
important in anaphylaxis in rats and mice but
its role in human beings is uncertain.
2 . ( i h t r m o t n u l i c f a c to r s : The eosinophil
chemotactic factors of anaphylaxis (E C F - A) are
acidic tetrapeprides released from mast cell
granules which are strongly chemotactic for
eosinophils. These probably contribute to the
casino philia accompanying many hyper­
sensitivity states. A high molecular weight
chemotactic factor has been identified, which
attracts neutrophils (NCF),
Heparin is an acidic mucopolysaccharide. It
contributes to anaphylaxis in dogs, but apparently
nor in human beings.
Enzymatic mediators such as proteases arid
bvdrolases are also released from mast cell
granules.
uiftaL^ai™ fit ••si staeME
1. Prostagland ins and Icukotriunes: They
are derived by two different pathways from
.Lrachidnnic pc id, which is formed trr>m
disrupted cell membranes of mast cells and other
leucocytes. The lipoxygenase pathway leads to
the formation of leuhotxienes, while the cyclo-
oxvgenuse pathwiv leads to prostaglandins and
thromboxane. A substance originally
demonstrated in lungs, producing slow,
sustained contraction of smooth muscles, and
therefore termed slow reacting substance of
anaphylaxis (bRS-A) has since been identified
as a family of Icukotriencs ilT B 4. C4, D4t E4).
Prostaglandin F2ri( and thromboxane A 2 are
powerful, but transient, bronchoconstrictors.
Prostaglandins also affect -secretion by mucous
glands, platelet adhesion, permeability and
dilatation of capillaries and (be pain threshold.
2 , P la te le t activ a tin g fa cto r ( P A F )t PAF
is a low molecular weight lipid released from
basophils which causes aggregation of platelets
and release of their vasoactive amines.
Besides
the products of masr cells and Other leucocytes,
several other biologically active substances have
been implicated in anaphylaxis. These include tile
anaphylatoxins- released by Complement aetivation
and bradykjiLLu and oilier kinins formed from plasma
kininngens.
I n tta v e n o u s
injection of peptone, tvpsin and certain other
substances provnhes a clinical reaction resembling
anaphylactic shock. Tliis is termed 'anaphylactoid
reaction’- The clinical resemblance is due to the
same chemical mediators participating in Hnth
reactions. The only difference is that anaphylactoid
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164 i Ts<rt>coi< of Mkcnsbioloqy ►
shock h:i:■■ no i m mu n o logi c a I basis and is a
nonspecific mechanism involving the activation o f
complement and the release ofaniphykiomiiis.
Atopy; T h e term 'atopy1 (Urerally m eaning nut
of place or strangeness) was introduced by Coca
! 1923) to refer to naturally occurring familial
hypersensitivities of human brings, typified by hay
fever and asthma. The antigens commonly involved
in atopy are characteristically inhalants {for
exam ple, pollen, house dust; or ingestartts (for
example, eggs, milk). Some of them are contact
allergens, to which the skin and conjunctiva may
be exposed. These atopens are generally not good
antigens when injected parcntcraJiy but induce IgE
antibodies, formerly termed as 'reapin' intihodies.
Atopic sensitisation is developed spontaneously
following natural contact with itopens. It is difficult
to induce atopy ariiiicially.
Predisposition to atopy is genetically determined,
probably linked Do\1HCgenotypes. AtOp^ therefore
runs in families. What is litherired is not sensitivity
to a particular antigen, or a particular atopic
syndrome but the tendency to produce IgE
antibodies in unusually large quantities. All
individuals arc capable of forming IgE antibodies
in small amounts but in atopies IgE response is
preponderant- About 10 per cent of persons have
this tendency to overproduce IgE. It has been
reported that borllefed infants tend to develop atopy
in later life more often than breastfed babies.
IgE'! differs from other Immunoglobulins i.n the
following respects;
1. It cannot be demonstrated bv the conventional
serological reactions such as precipitation or
complement fixation, 'lhe first in vitro method
for IgE detection was the radioallergosorbent
test (R A 5T ). Simpler techniques such as
ELtbA and passive agglutination have since
been introduced.
2. While atopy occurs commonly in human bci ngs,
it is not easy to induce it experimentally in
animals.
3. IgE is homocvtutropiL', that is, species specitic.
Only human IgE can fbt to the surface of human
cells. This is the basis of the Pransnitz-Kustner
(PK) reaction, which was the original method
for detecting atopic antibody. Prausnitz and
Kustner (1921) reported that if serum collected
from Kustner, who bad atopic hypersensitivity to
certain species of cooked fish, was injected
intracutaneoush into Prausn itz, followed 24 hours
later by an iiuracutaneous injection of a small
quantity of the conked fish anti^n into the same
sire, ,l whc.il iind ll.iR1 reaction occurred within a
few minutes. As tcagjnic IgE is homo--cytotropie>
the test has to be carried out on human skin. It
carries the risk of transmission o1 infection and so
is no longer used.
A. Unlike other antibodies, IgE is heat sensitive
and is inactivated at S6 PC in 2 -4 hours.
Hearing appears to damage die Fr part of the
IgE molecule, which is necessary for fixation
to cclls-
5- Atopic antibody docs nnt pass through the
placenta.
Atopic sensitivity is due to an overproduction
of IgE antibodies- This is often associated with a
deficiency of IgA. This association has led to the
suggestion that IgA deficiency may predispose to
atopy. The distribution of lymphocytes capable of
synthesising IgA and IgE is closely parallel,
especially m the submucosa. In normal indivi duals,
the inhalant and ingestant antigens are dealt with
by IgA lining the respiratory and intestinal mucosa
and therefore they do nut cuitie into von tact with
the potent id JgE producing cells. When IgA is
deficient, the antigens cause massive stimulation of
IgE forming cells, leading to overproduction of IgE.
The symptoms of atopy arc caused by the release
of pharmacologically active substances following
the combination of the antigen and the cell fixed
IgE. he clinical expression of atopic reactions is
usually determined by iiiu portal of entry uf the
antigen— conjunctivitis, rhinitis, gastrointestinal
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symptoms and dermatitis following exposure
through the eyes, respiratory tract, intestine or skin,
respectively. Sometimes the effects may he at sites
remote from the portal of entry, for example,
urticaria following ingestion of the aElcrgen.
Speciflc dcsensidsitiiin (hypOsensitiiiiiliuit) is often
practiced in die tntEment of atopy.
These reactions involve a combination of IgG (or
rarely IgM) antibodies with the antigenic
de ternai nan ts on the surface of cel In leading to
cytotoxic or cytolytic effects. Example# are lysis of
red cells caused by an fierythrueyie lotibodie* in
autoimmune anemias and hemolytic disease of the
newborn- AJtGrrtiliv'elv, a free antigen or hapten mair
lie absorbed on cell surfaces. Subsequent reaction
of the combined antigen or hapten with its
corresponding antibody leads to eel! damage. Many
drugs may act in this manner, leading to
complement mediated Lysis of red cells, leucocytes
and platelets, causing hemolytic anemia,
agranulocytosis and thrombocytopenic purpura.
In some Tvpe IT reactions, the antihndv
combines with cell surface receptors and disrupts
normal function, either by uncontrolled activation
(agoniat effect as caused by the ajifibixJv'long-aeting
thyroid stimulator" in Graves' disease) or by
blocking (antagonisteffect as in myasthenia gravis).
Arthus (1903) observed that
when rabbits were repeatedly injected
subcutaneously with normal horse scrimp the initial
inject to ns had no Local effect hut with Later
injections, there occurred intense local reaction
consisting of edema, induration ami hemorrhagic
necrosis. This is known as the Arthus reaction and
is a Local manifestation of generalised
hypersensitivity. The tissue damage is due to
formation of antigen-antibody precipitates causing
complement activation, and release of inflammatory
molecules. This leads to increased vascular
permeability and infiltration of the sire with
neutrophils. I -eueocyte—platelet thrombi are formed
that reduce the blood supply and Lead to tissue
necrosis. The Arthus reaction can be passively
transferred with fieffl con tain in g precipitating
antibodies (IgG-, IgM ) in high litres.
Arthus reaction terms a pathogenic Component
of many clinical syndromes. For example,
mtrapulmonary Arthus-like reaction to inhaled
antigens, such as thermophilic aetinomyeetesi from
mpuldy hay or grain causes Farmer's Jung and other
types of hypersensitivity pneumonitis.
This is a systemic form of
Type 111 hypersensitivity. As. originally described
by von Pirquet and Schick (19SJ5), this appeared
7 -1 2 days following a single injection of a high
concentration of foreign serum such as the
diphtheria antitoxin.The clinical syndrome consists
of fever, ly] up] Lade nopat hy, splenomegaly arthritis,
glomerulonephritis, endocarditis, vasculitis,
urticarial rashes, abdomin.il pain, nausea and
vomiting. The pathogenesis is the formation of
immune complexes (consisting of the foreign sc ruin
and antibody to it that reaches high enough litres
by 7 -1 2 days), which get deposited on the
endothelial Lining of blood vessels- in various parte
of rhe body, causing inflammatory infiltration.
The plasma concentration of' complement fails
due to massive complement activation and fixation
hy the antigen antibody complexes. The disease is
self limited. W ith continued rise in antibody
production, the immune complexes become larger
and uiotc susceptible to phagocytosis and immune
cliruination. When all foreign antigen is thus
eliminated and free antibody appears, the symptoms
clear without any sequelae. The latent period of 7 -
12 days is required only for serum sickness following
a single injection. With subsequent injections, the
disease manifests earlier. Scrum sickness differs
from other types of hypersensitivity reaction in that
a single injection can serve both as the sensitising
dose ami the shocking dose. As heterologous serum
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injections arc not used often now, the syndrome is
currently more commonly seen following injections
of penicillin or other antibiotics,
Immune complexes occur in many diseases,
including bacterial, viral and pmusltLC infoctluns (for
example, poststreptococcal glomerulonephritis,
hepatitis type U, m alaria), disseminated
malignancies and autoimmune conditions. The
nephritis and arthritis seen in these conditions may
be caused by deposition of immune completes,
Type IV hypersensitivity reactions (delayed
hypersensitivity) constitute one aspect ot cell medi ated
immune response. These are typically provoked by
intnaeelluljr niLLiKdud infections or haptens like simple
chemicals applied on the skin, evolve slowly and
consist of a mixed cellular reaction involving
lymphocytes and macrophages in particular. The
reaction is not induced by circulating antibodies bur
by sensitised T cells (Tdth,T hl,T h2, Tc) which, on
contact with the specific antigen, release cytokines
that cause biological effects on leucocytes,
macrophages and tissue cells, Delayed hyptsseiksitiviry
cannof be passively transferred by scrum but car be
transferred by lymphocytes of rite transfer factor. Two
types of delayed hypersensitivity are recognised —the
tuberculin (infection) type-and the contact dermatitis;
type.
The archetype
of delayed hypersensitivity is the tuberculin
reaction. When a small dose of tuberculin is injected
in tru derm illy in in individual sensitised to
tubcrculoprotc in by prior infection nr immun isati on,
an indurated inflammatory reaction develops at the
site within 40-72 hours, In unsensitised individuals,
the tuberculin injection provokes HOresponse. The
tuberculin test therefore provides useful indication
of the stare of delayed hypersensitivity (cell
mediated immunity) to the bacilli. The tuberculin
test differs from the skin test for Type 1
hypersensitivity nor only in the longer interval for
appearance but also in its morphology and
histology.
Tuberculin type hypersensitivity develops in
many infections with bacteria* fungi, viruses and
parodies, especially when the direction is subacute
or chronic and the pathogen intracellular. A similar
hypersensitivity is developed in allograft reaction
and in many autoimmune d b c-en
A
local reaction resembling the tuberculin response
may he produced by intradcrmal infection of some
protein antigens. This Is not a delayed
hypersensitivity reaction as it dan be passively
transferred by serum. Its histology is different from
the tuberculin response* being characterised by
jitomincnt basophil infiltration. This was formerly
known as the Joncs-M ote reaction but is now
termed cutaneous basophil hypersensitivity. Its
significance is not known.
Delayed
hypersensitivity sometimes results from skin contact
with a variety of chemicals—metals such as nickel
and chromium* ss-imple chemicals like dyes, picryl
cli loti tie, dinitrochlorobenvene, drugs such as
penicillin, find toiletries. Sensitisation is particularly
liable when contact is with an inflamed area of
skin and when the chemical is applied in an oily
base. Antibiotic ointments applied on patches of
dermatitis frequently provoke sensitisation. The
substances involved are in themselves not antigenic
but may acquire antigenicity on combination with
skin proteins. Sensitisation requires percutaneous
absorption. As most of the substances involved are
fat soluble, passage along sebaceous glands may be
the method of entry of tlie allergens.
Lange rhans’ cells of the skin capture locally
applied hapten, along with the modified tissue
proteins, and migrate to the draining lymph nodes
where they present the processed antigen along
with MHC molecules to T cells.The sensitised T
cells travel ro rhe skin site, where on contacting
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 * Hypomeraithrity ►
the an rigen they realeast var ii>us lymphoki ncs. Thl
cc IL* secrete TFNy and 11,3 winch activate
macrophages and other lymphocytes, Th2 cells
release IL 4 hIL>, G M -CSF and other factors that
lead to an influx of eosinophils and tissue damage.
Activated T c cells mediate killing of target cells,
Contact with the allergen in a sensitised
individual leads to 'contact dermatitis'* the lesions
varying from macules and papules to vesicles that
break down, leaving behind taw weeping areas
typical of acute eczematous dermatitis,
Hypersensi rivity i* detected by the 'patch test’. The
allergen is applied to the skin under an adherent
dressing. Scnsirivity is indicated by itching
appearing in 4 -5 hours* and local reaction which
may vary from erythema to vesicle or blister
formation, after 24-28 hours,
SHWARTZMAN REACTION
This is not an immune reaction but rather a
perturbation in factors affecting iintravascular
coagulation. It is traditionally described along w ith
hypersensitivity reactions because of a superficial
resemblance.
Shwartiman (1928) observed that if a culture
filtrate of S. ryphi is injected mcruderm,lIo in a
rabbit, followed 24 hours later hy the same filtrate
intravenously, a hemorrhagic necrotic lesion
develops at the kite of the iotradermal injection.
The intradcimal and intravenous injections need
not be of the same or even related endotoxins.
Culture suspensions or filtrates of a variety of
bacteria will Sensitise the skin to intravenous
injection by an equally wide variety of cultures or
filtrate*- This absence of specificity .Uid the short
interval between the two doses preclude any
immunological bans for the reaction.
The initial (preparatory) dose is
characteristically an endotoxin. The intravenous
{provocative] injection can be a variety of substances
— bacteri al e ndn tox ins, a nt igc n -an t ibn dy
Complexes, starch, scrum, kaolin and others. The
preparatory injection causes accumulation of
167
leucocytes which condition the site by release of
lysosomal enzymes damaging capillary walls,
Following the provocative dose, there occurs
intravascular clottings the thrombi leading to
necrosis of vessel walls and hemorrhage.
If both the injections are given intravenously,
the animal dies 12 2-1 hours after the second dose.
Autopsy showy bilateral corneal necrosis of the
kidneys and patchy hemorrhagic necrosis in the
Liver, s|jleen and othc r organs. An essent i.Jly simi lar
phenomenon was described by Sanarclli (1924) in
experimental cholera. The reaction is therefore
called the Sanarelli-Shwarezman reaction or the
generalised Shwartzman reaction.
It has been suggested that mechanli-ms similar
to the Shwartzman reaction may operate in some
clinical conditions such as the purpuric rashes of
meningococcal Sept ice m il and the acute
hemo r rh ag i.; adrenal necrosis found in
overwhelming infections (W aterh ouse-
Friderichsen syndrome).
Many infections, particularly Gram negative
septicemias can lead to a septic shock syndrome
with profound hypotension, hypoxu and oliguria.
This may sometimes be accompanied by the adult
respiratory distress syndrome (A R D S) with
overwhelming neutrophil invasion of lungs.
Massive activation of complement by the
alternative pathway, assoc:.ited with release of
thromboxane A2 and prostaglandins from platelets
may lead to disseminated intravascular coagulation.
The mechanism may be the excessive release of
cytokines such as the tumour necrosis, factor and
interleukins 1 and 6 by macrophages and endothelial
cells in response to -contact wii t h Li rgu qua.nl ities of
lipopolysaccharide endotoxin. Some Gram positive
infections may also cause similar effects. Staphylo­
coccus aureus can induce T N F secretion by
macrophages and peptidoglycan mediated platelet
aggregation, leading to disseminated intravascular
coagulation. Staphylococcal enherutux in can act i:-
a super antigen, activating whole fan -ilien of I" -cells
irrespective of their antigen specificities and causing
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168 i Textbook of Microbiology *

mwsiir'c rclfflsc of cytokines, leading to the toxic origin but their pathogenic mechanisms re-emtdf
shock syndrome. These orndir ions arc not of iinmune those In Iimmune mflamma:! ihi.

Further Rciilin|
QupcL H and M Hkiky 1993. Essenti--Is o f Clinical brnnunofogr. 3^edn. Oxford: Blockwr]].
Enin PW. 199*. AiuphyLui;, B M J31& .U 42.
Flunk M e( i] (eds). 1995- /mjrtunctJctLr-'cay D t-km ■■■v BoMon; I i'ile Brawn.
STl und M K Churth 1993- Mhfgy- \-tjndnTi; Guwcr
Howanh PI 1. 1990. AUtr^' : Pirhnfpmc mechanism. jVew F.ngj 343:712,
I,;k hin.iin: P et jl. 1993. ri■.■■'■rgy. OiJiiiil. Eil.L.'rtwII.
Q U iw C ll A x p tC S i ■•( J 11j.'ri:
Koitt JM. 1994. J . r . :.i.1hnummift^y. M' eiin. Oxford: filactwtlL

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 Autoimmunity
Self-antigens are not ordinarily immunogenic.
Ehrlich (19 0 1 ) observed that goats produced
antibodies against erythrocytes from other goats but
rot against their own, and postulated the concept
of 'horror Hiroroxicus’, But he did not regard
jutoi mmunif.iltion as an impossibility and even
envisaged it* pathogenic possibility.
Autoimmunity is a condition in which
ftriLcrui.il or functional damage is produced hi' the
action of immunologic ally competent cells or
antibodies against the normal components of the
body. Autoimmunity literally me min protection
against self' but it actually implies 'injury to self
and therefore it has been criticised as a contradiction
in terms. Autoallcrgy' has been suggested as an
acceptable alternative but the term autoimmunity
has the sanction of wide usage.
The earliest example of autoimmunity was the
olrserva tian by MeTiilrnkjofT(l90(» that guinea pigs
injected with their own spermatozoa produced
bperm immobilising antibodies. Donat h and
fandKreiner (1904) identified circulating
auroantibodies in paroxysmal cold hemoglobinuria
— a hemolysin which binds with the patient’s
crvlhrocvtcs at low temperatures and produces
complement dependent hemolysis on warming.
This Wifi the tirsi description of art autoimmune
disease in human beings, Datneshek and Schwartz
(1938) established the autoimmune basis of acute
hemolvtic anemia. With, the discovery o f Coombs
tc-HE for incompiete imtilwuLies it became possible
to demonstrate globulins bound to the surface ot
erythrocytes in this condition, Autoimmunisation
could be induced in exp erim ental animals by
injection of self-antigens along with the complete
Freund’s adjuvant. I'hc use of sensitive serological
techniques led to the demonstration of
autoantibodies in several diseases and even in a
proportion of healthy individuals. Some
auEoantihodies, such as the anti idiotypic antibody
may even be essential for the normal functioning
of the immune system.
When the concept of autoimmunity came to be
accepted as a pathogenic mechanism, a Large number
of diseases were suggested to have an autoimmune
etiology, based un the finding (it awlo.uiti bodies in
the patients. This was soon recognised to be
tin ten able as autoanfibodie* could be often
incidental or the result, and not the cause of disease.
Criteria were proposed for proving the
authenticity ot putative autoimmune diseases,
similar to Koctfs postulates in infectious diseases.
These were found to be not applicable in the case
of such spontaneous and multi factorial conditions
as autoimmune diseases, h may be proper to restrict
the rcrin ’autoimmune diseases' to those where
autoimmune processes, humoral or cellular, urc
shown to be responsible for the pathogenesis, rather
than merely associated. This is not strictly adhered
k>. Moreover, the border between autoimmunity and
hypersetuilivity is largely ill-defined or even
nonexistent.
Diseases of autoimmune origin usually exhibit
the following features;
1. An elevated level of immunoglobulins.
2. Demonstrable autnantibodies-
3- Deposition of immunoglobulins or their
derivatives at sites of election, Latch as renal
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170 * To <Ifoenit 0i MiOrOtiOlOfl/ ►
glomeruli.
4, Accumulation of lymphocytes and plasma ceils
at the sues of lesion.
5, Benefit from corticosteroid or other
immunosuppressive therapy.
6- The occurrence of more than one type of
autoimmune lesion in an individual.
7. A genetic predisposition towards autoimmunity.
ftr Incidence higher among females.
9, Chroniciry, Usually nonreversible.
MECHANISMS OF AUTOIMMUNISATION
Cells or tissues may undergo antigenic alteration
as a result of physical, chemical or biological
influences. Such altered or 'neoantigens1 may clidi
an immune response. Ncoantigicns can arhc in a
variety of ways. Physical agents such as irradiauon
can cause an rigen ic altera rii in, Photosensi rivity and
cold allergy may represent sensitisation to -.df-
antiigen.sn altered by light and cold, respectively.
Several chemicals, including drugs, can combine
with cells and tissues and alter their antigenic
nature. Contact dermatiLis, which is traditionally
considered a type of delayed hypemensnivity, can
al&o he taken to be an auto immune response to skin
ant igens altered by the ir comb ination wi th chcm ical
allergens. Drug induced anemias, leucopenias and
thrombocytopenias often have an autoimmune basis.
Infectious micnooigar-isms. particularly vimses and
other intracellularpathogens, may induce alteration
of cell antigens. Viral infections, such as infectious
mononucleosis, arc known to often precede
autoimmune diseases. Bacterial enzymes also induce
alteration of cell antigens. Neuraminidases formed
by myxoviruses and many bacteria act on
erythrocytes releasing the 1’ antigen. The almost
universal occurence of T agglutinins in human
sera is believed to represent a harmless autoimmune
response following infections, Ncoanugens may also
arise by mutation. Such mutant cells may he
immunogenic.
Immunological damage may result from
immune responses induced by cross-reacting
foreign antigens. The fortuitous similarity between
some foreign and self-antigens is the basis of the
'cross reacting antigen’ theory of autoimmunity.
Organ specific antigens are present in several
species. Ir'cctiion of heterologous oigan specific
antigens may induce an immune response damaging
the particular organ or tissue in the host. An a ample
is the neurological injury that used to be a
complication of anti:rahir immunisation in human
beings with the neural vaccine of infected sheep
brain tissue partially denatured by treatment with
phenol. Its injection elicits an immune response
against sheep brain antigens. This may cause
damage to the individual^ nerve tissue due to the
cross-reaction between human and sheep brain
antigens. Immunological injury due to cross­
reacting antigens can also follow infections.
Streptococcal M proteins and the heart muscle share
antigemc characteristic!;. The immune response
induced by repeated streptococcal infection can
therefore damage the heart. Nephritogenic strains
of streptococci possess antigens found in the renal
glomeruli. Infection with such strains may lead to
glomerulonephritis due to the antigenic sharing.
A related type of autoimmunisation is 'molecular
mimicry' which is due to the presence in some
infect mg microorganisms and self-antigens, of
eyitopes with identical peptide sequences {instead
of similarities in 'cross-reactions1). Examples of
such homologous sequences arc seen in
arihritogenil: Shigella flexneri and H L A j B27,
jVf>votacTcni rm mbcmitodis and joint membranes,
Coxsackle E and myocardium.
Another hypothesis is polyclonal B cell
activation- While an antigen generally activates only
its corresponding B cell, certain stimuli
nonspedfically cum on multifile B cell clones- Such
stimuli include chemicals (for example, 2-
mercaptoethanol), bacterial products (P P D ,
lipopoLywechaiideh enzymes (trypsin)* antibiotics
(nystatin) and infections with some bacteria
(mycoplasma), viruses (E B virus) and parasites
(malaria). Multiple nonspecific antibodies form
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during some infectious diseases, such as antihuman
erythrocyte cold, anti bodies in mytoplasm a
pneumonia and anti sheep erythrocyte antibody in
infectious mononucleosis.These polyclonal
antibodies are IgM in nature, similar to the "natural
antibodies’ produced by CD5^ B cells.
Breakdown of immunological homeostasis may
lesd to cessation of tolerance and the emergence of
forbidden clones of immunocompetent cells capable
of mounting immune response against scl (^antigens,
AutOLirmriunisation may result when tolerance ro a
selF-xn tigen is abrogated, as for instance by the
injection of the self-antigen with Freund's adjuvant.
Enhanced helper I cell and decreased
suppressorTcell functions have heen suggested as
causes of autoimmunity Defects in the thymus, in
stem cell development and macrophage (unction
have also heen postulated as causes.
Certain self-anti gens lire present in closed
systems and me not accessible to the immune
apparatus, These arc known as sequestered antigens.
An example is the lens antigen of the eye. The lens-
protein is enclosed in its capsule and. docs not
circulate in the blood.. Hence immunological
tolerance against this antigen is not established
during fetal life. When the antigen leaks out,
following penetrating injury, if may induce am
immune response causing damage to the lens of
the other eye. An example of "sequestration in time’
is seen with sperm antigens. As spermatozoa
develop only with puberty, the antigen cannot
induce tolc ranee dun ng letal life-JITc sperm antigen
is the reh >re not recognised as self and wbe tl it enters
the circulation, it is miniunogtidc, This is believed
to be the pathogenesis of orchitis following mumps.
The virus damages the basement membrane of
seminiferous tubules leading to the leakage of
sperms and initiation of an immune response
resulting in orchitis.
Defects in the idioitype-rantiidintype network
have also been said to lead to autoimmunity.
Genetic factors such as defective h or
immunoglobulin genes have also been postulated.
In human autoimmune diseases and in animal
models, genetic factors appear to influence the
development a nd fate of autoimmune states. In sp ite
of so many dirterent possible mechanisms, proposed,
their actual role in autoimmunity, if any, has not
been established,
Many an imul models- of spontaneous and induced
autoimmunity have contributed to an understanding
of this condition. Examples of spontaneous
autoimmune diseases in animals arc autoimmune
hemolytic anemia in the New Zealand Black
(NZR) mouse strain, systemic lupus erythematosus
in NZB X NZW cross, insulin dependent diabetes
mellirus in the nonobese diabetic {NOD) mouse,
and thyroiditis in the obese strain (OS) chicken.
Experimentally, autoimmunity car be induced in
many animal species by injecting tissue extracts in
the complete Freund's adjuvant~—for example,
experimental allergic cncephaluniyelitis with brain
or spinal cord extracts, and thyroiditis, with thyroid
gland extract.
Based on the sate of involvement and nature of
lesions, autoimmune diseases may be classified as
hemocytolylic, localised (or organ specific}, systemic
(or nonorgan specific), and transicorv diseases.
Auto antibodies against erythrocytes are
demonstrable in this condition. Serologically, two
groups of autoimmune anemias can be
distinguished, characterised by "cold" and 'warm’
antibodies, respectiveLy.
The cold autnantibodies aie, generally; complete
agglutinating antibodies belonging to the IgM class
and agglutinate erythrocytes at J "C but not at 37 "C.
Cold agglutinins were first detected by Doitath and
I.andsteiner in paroxramal cold hemoglobinuria.
This condition, which used to frequently
accompany syphilitic infection, is seldom seen
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nowa.dd.yH. Cold agglutinins art also seen in pnrn&ry
atypical pneumonia, trypanosomiasis-and blackvratcr
fever.
Warm autoantibodies are generally incomplete),
nonaggfe delating antibodies usually belonging to
the lgG clast. They can be shown coating the
erythrocytes in the direct Coombs test. Warm
anti erythrocyte antibodies are frequently seen in
patients, taking certain drugs such as SLilphonamides,,
antibiotics, and alpha methyl dopa,
In autoimmune anemias, the red cells coated
with antibodies are prematurely destroyed in the
spleen and liver. Complement dependent
intravascular hemolysis appears lo be a rare event.
Ajurxxanubodies directed against platelets occur in
idiopathic thrombocytopenic purpura. SedoJTnid
purpura is an instance of immune response against
drug induced neoantigens on platelets. This
condirioit is traditionally considered an antibody
mediated hypersensitivity.
Nonagglutinating
antileucocytc antibodies can be demonstrated in the
scrum of patients with systemic lupus
erythematosus and rheumatoid arthritis.
1 . M nshim nro's d ise a se (L ym p h ad en o id
gmLrek T h is is the most typical ami best
studied of organ-specific autoimmune diseases.
Iti 1 9 5 6 P Koitt and Doniaeh in England
demonstrated arltithyrOglobulin antibodies in
the sera of patients by precipitation in gel, and
Witcbsky and Rose in the USA by the more
sensitive passive hemagglutination lest. The
latter workers also reproduced the disease in
rabbits by immunisation with autologous thyroid
tissue obtained by hemichyroidectomy.
Hashimoto1!; disease occurs more frequently in
females and is associated with an enlargement of
the thyroid gland mid symptoms of hypothyroidism
or trank myxedema. Histologically the glandular
structure is replaced by lymphoid tissue consisting
ot lymphocytes* histLocytes and plasma cells.
Antibodies with different specificities have been
found in this condition. They include antibodies
that react with thyroglobulln, a second acinar
colloid, microsomal antigen and a thyroid cell
surface component.
2 . T h y ro to x ic o s is (G ra v e s ' disease)* The
majority of patients with thyrotoxicosis possess
an tib od y to th yroglob u lin . L y m p h o cy tic
infiltration is common in thyrotoxic glands. 'I'he
immunological basis of thyrotoxicosis is
supported by the identification of the 'long
acting thyroid stiniulitoF (LATS) which is an
IgC antibody to the thyroid membrane antigen.
Combination of I.ATS with the surface
membrane of thyroid cells seem?; to stimulate
excessive hormone secretion.
The immunological basin
of Addison’s disease is suggested by lymphocytic
infiltration of the adrenal glands and the presence
of circulating antibodies directed against the cells
of the zona glomerulosa. Similar lesions can be
produced in experimental animals by immunisation
with adrenal tissue in Freund’s adjuvant.
Experimental allergic
orchitis writh progressive damage to germinal
epithelium and aspermatogenesis. can be induced
in guinea pigs by the injection of autogenous or
allogeneic testes with Freund's adjuvant. A similar
condition so me times follows mumps orchitis.
Lymphocytic in frit rat ion of the t cites and
circulating antibodies tn the Sperms and germinal
cells can be demonstrated in this condition.
In this disease, there is
an abnormal fat in ab ility of muscles due to
malfunction of the myoneural junction. An antibody
against acetyl choline receptor on myoneural
junctions of striated muscles is present in these
patients. This prevents acetyl choline from
combining with its receptor, and impairs muscular
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contraction. The thymus shows lymphoid
hyperplasia and numerous germinal centres. Jnfonts
bor [] io affected mothers show symptoms of the
disease hut rtcovtt spontaneously hy the jge of two
months, coinciding with the disappearance of
maternal antibodies. This suggests that the
pathogenic factor in neonatal myasthenia mav be
the autoantibody passively acquired from the
mothe r.
Two
types of autoimmune diseases arc seer in the eyc-
C a tin ct surgery sometimes leads to intraocular
inflammation caused hy the autoimmune response
to the lens protein. This is known as
pha ma naphyisxis.
Perforating injuries of rhe eye, particularly those
i nvohi ng the iris or ci liary bodies a re often followed
by ffinp^rhetrc ophEhaiirfa in the opposite eye.The
disease can be produced in rxperimental animals
by immunisation with uveal or retinal tissue in
Freunds adjuvant and can he passively transferred
with the spleen or lymph node cells but not wkh
serum.
Two types of
autoanrihodies are present in this condition. The
first is directed against the parietal cells of the
gastric mucosa. This is believed to cause
achlorhydria and atrophic gas [rids. The second type
of antibody is directed against the intrinsic factor
and prevents absorption of vitamin Bu eirhet by
blocking its attachment to the gastric intrinsic factor
or by binding to the ft|j: intrinsic factor complex
and interfering with its uptake hy the intestinal
mucosa.
The 'neuroparalytic accidents' following
rabies vaccination represent injury to the nervous
system hy the immune response against the sheep
nervous tissue in the vaccine, which cross reacts
with human nerve tissue. An essentially similar
condition, experimental alleiglc encephalomyelitis
(E A E )t can be produced in animals by
immunisation with nervous tissue in Freund's
adjuvant. The encephulogenic protein has been
identified as the myelin basic protein (MRP) which
shows no species specificity.
Idiopathic polyneuritis (Guillirin—Rirre
syndrome) is considered an autoimmune response
against the peripheral nervous tissue. It can be
reproduced in experimental animals by
immunisation with peripheral nervous tissue in an
adjuvant.
Three
serious diseases of the skin are considered ro
have an autoimmune basis.. Pemphigus vulgaris
may he caused hy an antibody tn the intercellular
cement substance. In bullous pemphigoid,
antibodies directed against the dermal epithelial
junction have been demonstrated. Specific
antibodies in dermatitis herpetiformis have not been
identified.
This group includes conditions characterised by
immune response against a variety of self-antigens
at id damage to several organs and tissue systems.
Klemperer ( 1942) classified a numher of diseases
of unknown origin with the common feature of
connective tissue lesions as 'collagen diseases’.
Ineluded in this category are systemic lupus
erythematosus. (S L E ), rheumatoid arthritis,
polyarteritis nodosa, Sjogren's syndrome,
dermatomyosiiis and scleroderma. All these
conditions are associated with generalised
autoimmune processes.
This is s
chronic, multisystem disease with remissions and
exacerbations, terminating fatally. Patients have a
variety of autoanrihudjes directed against Cell nuclei,
infra cytoplasmic cell constituent!;, immu.no
giahulins, thyroid and other organ specific antigens,
Biological false positive reaction is scon in standard
tests for syphilis. The abundance and variety of
au roantibodies suggest a breakdown in tine central
control of immunological homeostasis.
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 174 < Textbook of Microbiology ►
The first immunological feature identified in
SLE was the L E ceil phenomenon described in
1948. The L E cell is a neutrophil containing a
large* pale, homogeneous body {L E bod}) almost
fill mg the cytoplasm, [lie L E body is the
immurologically damaged nucleus of a leucocyte.
Sometimes, instead of being intracellular* the LE
body can be seen free, surrounded by a rosette of
neutrophils. The fact that L E cell formation is due
to an antibody fL E factor) present in SLE can be
demonstrated by incubating normal blood with
serum from an SLE patient. The nuclei of some
leucuCytes can be seen to swell and become pale
and spherical- Neutrophils can he observed to
surround these damaged cells, strip away the
cytoplasm and c:rq;i:ll the free rmuteus to form L E
cells. Oiiemsa stained smears of blood or bone
marrow can demonstrate L E cells, but its sensitivity
is so low that this test has been replaced by other
antibody tests for diagnosis.
Im m unofluorcscert tests for antinuclear
antibody {ANA) show up different patterns of
staining* such as homogeneous [diffuse), peripheral
{outline), speckled and nucleolar staining patterns.
ANA tests are sensitive but not specific for SLE*
as they may be positive in many other autoimmune
conditions, viral infections, chronic inflammatory
processes, as well as in persons using certain
medicines and in the aged.
Anti-DNA antibodies are tested by R1A or
ELISA, Three major types of these antibodies arc
Seen-1—those reacting with single stranded {ss),
double stranded (ds) and both ss and ds DNA. O f
these, high titre anti-ds DNA antibody is relatively
specific for SLE. Another SLE specific antilody
is anti-sm antibody.
R h eu m ato id a r th r iti c This is a symmetric
polyarthnin with muscle wasting and subcutaneous
nodules, commonly associated with scrositls,
myocarditis, vasculitis and other disseminated
It;-1,ms. Ir i.s found more commonly in women.The
synovial membranes of the affected mines are swollen
and edematous, with dense infiltration of
Lymphocytes and plasma cells. A striking feature is
the presence of a circulating auioantitaidy called
the 'Rheumatoid factor’ (RE). This is usually a 19s
IgM* though IgG* and IgA R F have also been
demonstrated. RF acts as an antibody ag.iiinsr the
Fc fragment of immunoglobulins. They combine
usually with IgG though some types of RF are
d:.netted towards other immunoglobulin classes, RF
reacts with autologous, i■iologpus or heterologous
immunoglobulins. RE is generally considered to
be an immunoglobulin behaving as anti body to
determinants present in the patient’s own l^( i
molecules, though some configurational alteration
of Igf! may be required before its reactivity with
RF becomes demonstrable.
RF is detected by agglutinat^m tests u^ing* as
antigens* particles coated with globulins. In the
R ose-Waaler tear, the original technique for
detection of RF, sheep erythrocytes coated with a
subaggluiin&nii.g dose of anti erythrocyte antibody
{amboceptor) are used as the antigen in an
agglut ina rion test. In modifications of the test, latex
and bentonite are used as the earner particles for
lgG. Ammudear antibodies are frequently found
in rheumatoid nrforirb.
P o ly a rte ritis n o d o ta : Thiv is a necrotizing
angiitis involving medium sized arteries, ending
fatally due to coronary thro mhos is, cerebral
hemorrhage or gastroi ntcsr i n,al bleeding.
Polyarteritis is seen as a component of serum
MLlcness and other tome complex diseases. Immune
complexes of hepariris B vii-us antigen (Hbs Ag) in
affected tissues* including the kidneys, have been
demonstrated in 50—40 per cent of pa:i L-nts, Hi i
it has beer suggested that polyarteritis nodosa may
be an autoimmune disease, the autoantibody
responsible has not been identified.
S jogren syn d ro m e; T hk is a triad of
cotjiuncm ids sicca* dryness of the mouth, with or
wilhout saliraiy gland ehLaigement, ard rheumatoid
artbri rk The syndrome may occur in association with
other collagen diseases. Antinuclear antibodies and
rheumatoid (actor commonly occur m sera.
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These include conditions Ktich as anemia.
thrombocytopenia Dr nephritis that follow ccrt&in
infections or drug therapy. The infecting agent or
drug induces antigenic alteration in some self-
antigens. The immune response set up causes tissue
damage. The disease is transient and undergoes
spontaneous cure when the infection is controlled
or the drug withdrawn.
Many diseases are considered to be o f autoimmune
origin, based on their association with cellular or
humoral immune responses against self-antigens.
Autoantibodics are more easily detected than cellular
autosensitisation. However, the mere presence of
autoantihodies during the course of a disease does
not prove their etiological role. Autoantibody
formation may bo a result of tissue injury and the
antibody may help in promoting i m m u n e
elimination of the damaged cell or tissue elem ents-
A typical example is lepromatous leprosy in which
^ tir DM and J Siewari 1997. lminunatcsgy. ft,h«iln. Edinburgh: Chmdiill Liviri|$stcme.
F’fcakjr.m M and D Ver^tii 1997. S hjjc anJ Cl. meal Immunology. Edinburgh: Churchill Livingstone.
Roin J ct al. 1999. Immunology. 5 ' edn. London: Musby.
large amounts of autwntiibodi cs arc regularly found,
k has been said that but for the lepra bacillus,
lepromatnus leprojy may have been proposed as an
autoimmune disease.
The relative importance of humoral and cellular
immune processes in rhe etiology of autoimmune
diseases is not known. Antibodies may cause damage
by the cytolytic or cytotoxic [type 2) and toxic
complex (type 3) reactions. They are obviously
important in hcmocytolytic autoimmune diseases.
Another mechanism of autoimmune tissue damage
is by sensitised T lymphocytes (type 4 reaction). It
is likely that humoral and cellular immune
responses may act synergistically in the production
o f some autoimmune diseases. For example,
experimental orchitis cao he induced only when
both types of immure responses are operative.
Once initiated, most autoimmune responses tend
to be self perpetuating. Theit progress can be
arrested by immunosuppressive therapy, though the
degree of response to such therapy varies in different
diseases.
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 Immunology of Transplantation and
Malignancy
When* as a result of disease or injury* an organ nr
tissue becomes irreparably damaged* or when an
organ is congenitally defective or ahsem,
transplantation or grafting becomes necessary for
tin; ntiUjiation of function. The tisiUt or organ
transplanted i* known as the transplant or graft.
The individual from whom the Era ohplant in
obtained is known as the donor and the Individual
to whom ii is applied, the reiipienr.
Transplantation Is one of mankind's ancknt
dreams-. Chimeras, fanciful creatures composed (it
parts from different species, figure in tire mythology
flnd pantheon of alt ancient nations. Such
transplantation* across the specie* barrier, however,
do not succeed. It was recognised very early that
transplants survive onlv when the tissue or organ
is taken from the recipient himself while grafts
from another individual of the same species of from
n different species would he rejected- ITic earliest
application of transplantation appears to have been
skin grafting for reconstruction of the severed nose*
using the patient’s uwn skin fluji-s - a teclLnic|ue
described in the Sushnitu Samhita (circa ECO BC).
11 ic reasons for the rejection of exogenous grafts
were for long suspected to he due to active immunity
hut it was only in the 1940s that the work of
Medawar and his colleagues conclusively proved
its immunological basis.
Transplants may he clarified in various ways:
1. Based on the organ or tissue transplanted, they
are classified as kidney, heart, skin transplant
and so on.
2 . Based on the anatomical sice of origin of the
transplant and the site of its placement, grafts
are classified as 'orthotopic1 and H heterotopia
Orfhotopic grafts are applied in anatomically
Lminuid' si res, as in skin grafts. 11 ctenotopic grafts
are placed in anatomically abnormal' sites* is
when thyroid tissue is transplanted in a
subcutaneous pocket.
3. Transplants may be of fresh tissues and organs
or of stoned ones.
4. Transplants may be of living or dead materials.
Live grafts, such as kidney or heart, are expected
to survive and function physiologically in the
recipient and are called H vital grafts'. Nonliving
transplants like bone or artery merely provide a
scaffolding on winch new tissue is Laid by the
recipient. They arc called ‘static1 or ‘structural’
grafts.
5. Transplants may be classified based on the
genetic (and antigenic) relationship between the
donor and the recipient (Table 20.1). An oigau
or tissue taken from an individual and grafted
on himself is an autograft. A graft taken from
;in individual and placed on another individual
of the same genetic constitution is called an
isugraft. drafts made between identical twins
or between syngeneic members of highly inbred
strains of animals are examples of isografts*
Grafts between rwo genetically non identical
members of the same species- are called allografts
(formerly called bomografts). Grafts between
members of different species arc called
xenografts (turner Ly called heterografts).
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When a skin graft from an animal (such as a. rabbit)
is applied on a genetically unrelated animal of the
same species* the graft appears to be accepted
initially. The graft is vasculiriscd and seems
morphologically and functionally healthy during
the first two or three days. Huwever, by about the
fourth day, inflammation becomes evident and the
graft is invaded by lymphocytes and macrophages*
The blood vessels within the graft are occluded by
thrombi* the vascularity diminishes and the graft
undergoes ischemic necrosis. With extending
necrosis* the graft assumes a scab-Like appearance
and sloughs off by the tenth day. This sequence of
events resulting in the rejection of the allograft is
known is the first set response {also known as the
first set rejection or reaction’).
Ift in an animal which has rejected a graft by
the first set response* another graft from the same
donor is applied, it will be rejected in an accelerated
fashion. VaKulariUlion commences but ls soon
interrupted by the inflammatory response. Necrosis
sets in early and the graft sloughs off by the sixth
day. The accelerated allograft rejection is known as
the second wf response*
The
immunological basis of graft rejection is evident
from the specificity of the second set response.
Accelerated rejection in seen only if the second graft
is from the same donor as the first. Application of
a skin graft from another donor will evoke only
the first set response.
Donor
Self
Different individual, genetically identical
with recipient. Identical twin or member ftf
inbred strain
Genetically unrelated member of same
species
Different species
An allograft will be accepted if the animal is
rendered immunolagically tolerant. If suitable living
celts (such as splenic cells) from one pure line strain
of animal are injected into fetal or neonatal animals
of another inbred strain, the latter, when they grew
up, will accept grafts from the former animal This
is due to the induction of specific immunological
tolerance against the donor tissues as a result of
contact with them during embryonic life. The
tolerance can be abolished by injecting lvtnpbocytes
from a nontolcrant syngeneic animal, or more
effectively, from a syngeneic animal sensitised
against the donor tissues by a prior
allotransplantation. This method of transferring
immunity by means of lymphoid cells is known as
adoptive immunisation.
Transplantation immunity is predominantly cell
mediated!* The first set response is brought about
almost exclusively by T lymphocytes. Humoral
antibodies, are also produced during allograft
rejection. They can be detected by a variety of
methods including hemagglutination,
lymphocyte toxicity, complement fixation and
immunofluorescence. Antibodies are formed more
rapidly and abundantly during a second SCI response
than during primary rejection. Antibodies are
believed to participate in the second set response
along with cell mediated immunity* When a graft
is applied to an animal possessing the specific
antibodies in high titres, hyperacute rejection takes
place. The graft remains pale and is rejected within
hours without even an attempt at vascukrisation,
JVtmt Sjm onytns
Autograft Autogenous or autogenic graft.
Isografr Isologous or syngeneic graft or
sj-ngraft
Allograft Allogeneic graft. Formerly called
hnmograft
Xenograft Xenogeneic. Formerly called
heterograft
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 173 * 7e?xlhaui of Microbiology *
This is known as the ‘white grift response'. This
type or hyperacute rejection is sometimes seen in
human recipients of kidney transplants, who may
possess preexisting antibody as a result of prior
truftsplinEaiion, transfusion or pregnancy. The
glomeruli in such cases arc choked by platelet and
leucocyte agglomerates. Donor specific blood
transfusions to recipients before kidney Transplants
have been found to favour graft survival. This may
he due to the enhancing effect of antibodies to
mismatched donor antigens, induced by the
transfusion.
Humoral antibodies may someilines act in
apposition ta cell mediated immunity, by inhibiting
graft rejection. This phenomenon, called
im munological enhancement was originally
described hy Kabss in tumour transplants. If the
recipient is pnetreated with one or more injections
of killed donor [issue and the transplant applied
subsequently, it survives much longer than in
control animals. The enhancing effect can be
passively transferred to normal animals by an
injection oT serum from immunised animals,
showing that the effect is due 10 humoral an l11'otbes.
The antibodies may bring about the cnhanci ng effect
in various ways. They may combine with the
antigens released from the graft so that they arc
unable to initiate an immune response {afferent
inhibition). The antibodies may combine with the
lymphoid cells of appropriate specificity and, by a
negative feedback influence, render them incapable
of responding to the antigens of the graft {central
inhibition). They may also cause 'efferent
inhibition* by coating the surface of cells in the
graft so that sensitised lymphocytes are kept out of
contact with them.
Allograft immunity 1 - a generalised response
directed against all the antigens of the donor. A
recLiuent sensitised hy a skm graft will reir'et hy the
second set response not only another skin graft but
also any other organ or tissue graft from the same
donor.
11 1 sri o m p a T i b i u t t ArJTic^EnJS
Immune response against transplants depends on
the presence in the grafted tissue of antigens that
are absent in the recipient and hence recognised
as foreign. It follow^, therefore, that if the
recipient possesses all ike antigens present in the
graft, there will he no immune response, and
consequently no graft rejection, even when the
donor and recipient art not syngentif. The first
generation {F l ) hybrids between two inbred
strains possess antigens representative of both the
parent strains .me I will therefore accept grafts from
cither of the parental strains. If the two parental
strains have genotypes AA and BB, respectively
the Fl hybrid will be of genotype AB. It can
therefore accept [issues with genotype AA as well
as BB, as it possesses both alleles. Transplantation
in the reverse direction (from F l to patent) will
not succeed as str^ in AA wi 11react against antigen
B and strain BB against antigen A.
While transplants between members of a
highly inbred strain of animals are successful, au
exceptior is seen when the donor is a male and
the recipient a female. Such grafts are rejected a&
the grafted male (issue (XY) will have antigens
determined by the Y chromosome which will be
absent in the female (XX) recipient. Grafts from
the female to the male will succeed- This
unilateral sex linked li isti>i nc<>mparibility is known
as the Eichwald-Sifmser effect.
Antigens that participate in graft rejection are
called transplantation or histocompatibility
antigens. The blood group anti gens air important
in transplantation. The term 'major
histocompatibility system1 refers to a system of
cell antigens that exert a decisive influence on
the fate of allografts. Major histocompatibility
systems have been identified in different species
- } 12 in mice, AgB in rats, B in chickens, HI in
rabbits and D LA in dogs. The major
histocompatibility system in human beings is the
human leucocyte antigen (H L A ) system. A
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description of the HLA system is presented in
Chapter IS.
1- Next to ABO blood group compatibility the most
important tactor ml allograft Survival is H I .A
Lonipatihilitv. This is tested by HLA typing anti
tissue matching. H L A typing identifies the
11 LA antigens expressed on the surface of
leucocytes.
The standard typing method is the microcyta
tqaddly echi. Lymphocyte suspensions ans added to
microwells of tissue typing trays predispensed wirh
a pane! of H LA typing sera, each containing
alloantibodies to » specific HLA antigen, and
incubated with complement. Cells carrying antigens
corresponding to the IJLA antiserum arc killed by
complement, mediated membrane damage. Tlicsc
can he detected by the addition of eosin or trypan
hluc which stains only dead cells. The Lymphocyte
js presumed to have IILA antigens corresponding
to the specificities of all the antisera that hive
caused cell death, as indicated by the staining.
Antisera for H LA typing were originally
obtained from Lnultigravidae, placental fluid and
In mi multi pie blood transfusion recipients, who
have antibodies against mismatched paternal nr
donor 11 LA antigens .These arc now being replaced
by monoclonal antibodies.
More discriminating molecular method-s have
been developed for tissue typing. These include
restriction fragment length polymorphism (RFLP)
with southern blotting, and polymerase chain
reaction (P C R ) amplification using sequence
specific primers.
Once a set of H LA compatible donors is
available (commonly, siblings of the patient), the
best donors among them can he chosen by tissue
matching. This is done by the mixed lymphocyte
reaction or culture ( M L R , M I .C ) . It depends on
the fact thatT lymphocytes in culture, when exposed
to HLA Incompatible antigens, will undergo blase
transformation, the intensity of the reaction being
a measure of the antigenic disparity between the
donor and recipient lymphocytes. The test, as
performed, is a one-way rest in which the donor
lymphocytes are killed and only the recipient
lymphocytes ire permitted to be transformed in
response to the incompatible antigens on the donor
cells,
2. As allograft rejection is an immunological
process, jmmujiosuppfrasfon will inhibit it, This
can be achieved in experimental animals by
neonatal thymectomy, chronic lymphatic
drainage or administration of ALS— procedures
that will inhihit ceil mediated immunity.
Clink'd Tmn;pkntudorj employs a combination
of immunosuppressive drugs, including steroids,
azathioprene and the fungal metabolite
cyclosporin A, which is currently the most
effective agent,
3, There appear to be certain privileged sites where
aElografts are permitted to survive, safe from
Immunological attack The fetus can be
considered an intrauterine allograft as it contains
antigens which aTe foreign to tlie mother. The
reason why the fetus is exempt front rejection is
not clear, though many explanations have been
offered. The placenta acta an an immunological
barrier by generating a hormone which is locally
immunosuppressive- Major histocompatibility
complex (M H C) antigens are present only in
low density on trophoblastic cel In and the cell
membranes are relatively resistant to attack by
T ur K cells. Antigeo shedding by the fetus
blocks the aggressive T cells or antibodies by
an enhancement effect. An incomplete
mucopolysaccharide barrier rich in sialic acid
surrounds the trophoblastic cells, protecting
them from cytotoxic lymphocytes. The high
concentration of alphafctoprotein m fetal blood
also maybe a factor, as it has immunosuppressive
properties, which may protect the fetus against
immunologic^] damage from any maternal
leucocytes entering fetal circulation.
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 An} site that is im penetrable to
immunocompetent cells (for example, cartilage) is
an immunologically privileged site.. Areas where a
lymphatic drainage system is absent such as brain,
hamster cheek pouch or ineffective such as testes
can accept allografts without rejection, Lack oi
oscular] ty at the site also prevents graft rejection.
This is the reason for the success of corneal
transplants.
Graft rejection in due to the reaction of the host to
the grafted tissue (host-versus-graft response). The
contrary situation., in which the graft mounts an
immune response against the antigens of the host,
is known as the graft-uersus-hosi (GVH) reaction.
The GVH reaction occurs when the following
conditions arc present:
1. The grift contains immunocompetent T cells.
2- The recipient possesses transplantation antigens
that ate absent in the giaft-
3. The recipient must not reject the graft.
Examples of situations leading to the GVH reaction
are:
a. Allograft in a recipient In whom specific
immunological tolerance has been induced.
b, Adult lymphocytes injected into an
immunologic ally deficient recipient. The
immunological deficiency may be due to
immaturity (newborn) or immunosuppression.
C- F, hybrid receiving a transplant from any one
parental strain.
The major clinical features of the GVTH reaction
in animals sue retardation of growth, emaciation,
diarrhea, hepatosplenomcgaly, Lymphoid atrophy
and anemia, terminating fatally, The syndrome has
been called runt disease.
When a cell undergoes malignant transformation, it
acquires new surface antigens. It may also lose some
normal antigens. This makes a tumour antig^nicaUy
different from the normal tissues oi die host. A tumour
can, therefore, he considered an allograft and he
expected to induce an immune response.
Several clinical observations indicate the presence
of an immune response that prevents, arrests and
occasionally cures malignancies.
1. Instances of spontaneous regression of established
tumours have been reported, especially with
neuroblastoma and malignant melanoma. On
the analogy of the role pLsytd by the immune
response in recovery from infections, it is
believed that recovery from malignancy also may
represent an immune process,
2. Dramatic cures sometimes follow chemotherapy
of choriocarcinoma and Burkitt's lymphoma.
Even a single dwe of cytotoxic drug may, on
occasion, result in a complete cure. Ag.iio, in
some types of tumours, such is hypernephroma
with pulmonary metastaaes, removal of the
primary tumour often leads to a regression of
the n K tistu s. These observations suggest that
once a large mass of tumour has been removed,
mopping up operations can be effected by the
immune process. The immune response appears
to be effective only when the tumour is below a
“Critica.] mass'.
3. There is a higher prevalence of certain types of
Cancers observed unexpectedly at autopsy,, than
their clinical incidence would suggest. This
indicates rhat the immune system is able to deal
with malignant cells as they arise and that only
some of them are able to overcome the defence
mechanisms and develop into clinical cancer.
4. Histological evidence of immune response
against malignancy is provided by the presence
of lymphocytes, plasma cells and macrophages
infiltrating tumours. The cellular response
resembles that seen in the allograft reaction.
Tumours shewing such cellular infiltration have
a better prognosis than those that do not.
5. If the immune system plays a natural role in
Copyrighted material
 < Immunology oF Transplaniaiion and Ma'ignancy ►
preventing u m a u r developm ent, a high
incidence of malignancy should be expected in
immune deficiency states.This is indeed so. An
increased incidence or cancer, parriculariy
lymph ore titular malignancies, is found in
congenital immunodeficiency suites, m AIDS
and in patients undergoing chronic
immunosuppressive therapy,
Tc u ou u A n tig en s
T u m o u r andijens are antigens that are present in
malignant cells but absent i,n the corresponding
normal cells, of the host.
Tumour specific antigens art present on the
membranes of malignant cells and induce an
immune response when the tumour is transplanted
in syngeneic animals. Such rumour specific antigens
which induce rejection o f rumour transplants in
immunised hosts arc termed tumour specific
transplant a cion antigens (T S T A ) or tumour
associated transplantation antigens {TATA).
In chemically induced tumours, the TSTA is
tumour specific. Different tumours possess different
T S [’A, even though induced by the same
car.Tiogen. In contrast, the TSTA o f virus induced
tumours is virus specific in thac all tumours
produced by one virus will possess the same antigen,
ever ]f the tumoura occur in different an imil strai ns
or species.
A second type of antigen is found in some
tumours. These are the fetal antigens which ate
found :n embryonic and ntuugnant cells but not in
normal adult cells. The best known examples are
alphafctoprotci n in hepatom as and the
carcinoembryonlc antigen found in colonic cancers.
Their synthesis represents a dedifferenti.LtJon of
malignant colls into more primitive forms.
The carcinoembryoriK antigen is a glycoprotein
wh ich can be detected in the serum of many pat ients
with carcinoma of the colon, particularly in the
presence of metastaScE-. However, it also appears in
some other conditions such as alcoholic cirrhosis,
and hence irs diagnostic value i-. lim ited.
181
AJphafetOprutei.it is an aJphaglobulin secreted by
normal embryonic hepatocytes, Its serum level drops
sharply after birth and is hardly detectable in adults.
High levels arc present in hepatic carcinoma, in
which condition ir is of diagnostic value, Prostate-
spediic antigen (PSA) has been used as a dignostie
indicator for prostate cancer.
iM H U N fi R b &p d n s & cn M a l ig n a n c h
Both humoral and cellular responses can be
demonstrated in malignancy. Anti-TSTA andbodics
can be dem onstrated by indirect m embrane
immunofluorescence. Delayed hype sensitivity to
tumour anti gens can be detected by skin testing with
tumour cdE extracts. Cell mediated immunity can
be demonstrated by the stimulation o f D N A
synthesis and lymphokinc production by the
patients leucocytes on exposure to the tumour
andgens. The lymphocytes from the patients are
cytotoxic to the Cultured tumour cells.
Cell mediated immunity is believed to be the '
mechanism of host defence against malignancy. The
humoral response may not be relevant, or miY even
be detri mental due to its fecilita ting tumour growth
by the process of enhancement.
Im m u n o lo g ic Ai S uhvelli -x n l : i -
The concept of immunolo^iral surveillance had its
beginning in the observations of Ehrlich (1906).
It was revived by Lewis TTiocnas in the 195% and
developed by Burner. It postulates that the primary
function o f cell mediated immunity is to 'seek and
destroyf malignant cells that arise by somatic
mutation. Such malignant mutations are believed
to occur frequently and would develop into tumours
but for the constant vigilance of the immune system.
Inefficiency of the surveillance mechanism, either
as a result of ageing or in congenital or acquired
immunodeficiencies, leads loan increased incidence
of cancer. W h Je this hypothesis is attractive, it may
perhaps represent an oversim p lification of a
complex ^ituation.
I f immunological surveillance is effeedve, cancer
C o p y rig h te d m aterial
1S2 s lH^ri&ook of M crebiatofly ►
should not occur. The development o f tumours
represents a lapse; in sur veil Lance. The mechan]---rrLs
of such lapses are not dear but several possibilities
have been suggested. Due to the very fast rate o f
proliferation of malii^iant celLs, thev nmv be able
to 'sneak through' before the development of an
effective immune response and once they reach a
Certain mass may be beyond the pow er o f
immunological attack. Circulating tumour andgens
may act as a ‘sinokescreen', cosiLng the lymphoid
cells and preventing them from acting on the
tumour cells. The rumour antigens on malignant
cells may be inaccessible to sensitised cells, being
covered by some antigenically neutral substance.
Humoral antibodies may cauie immunological
enhancem ent, "Blocking" activity has been
demonstrated in humoral factors. This may he due
to the circulating auLigen., an Libody or antigen-
antibody complexes. Some rumours may be of low
im m unogeniclry or may form cytokines like
transforming growth factor p (T G F -(il which
suppresses cell mediated i mmunity.
I mmi n o t h e r a p y in- C a n c e r
Different approaches have been attempted in the
immunotherapy o f cancer— passive, aclive and
adoptive immunotherapy, specific and nn nspeci lic.
Passive immunotherapy was the earliest method
of cancer immunotherapy. Antisera prepared by
immu nising an imils wi th tumour b!opsy specimens
were used for the treatment of human cancer .is
early as in 1995. This method was found to be of
no use and therefore abandoned. A special type nf
serotherapy has recently been found bereficiil in
experimental tumours. Appropriate antisera that
possess "deblockj ng1ac Livily in vitro have been found
IO Cause regression of tumours, apparently hv
neutralising the circulating tumour antigens and
permitting the sensitised lymphocytes to act cm
tumour cells. Monoclonal antibodies to tumour
antigens may play a role 35 Carrier? in transporting
Cytotoxic or radioactive drugs specifically to the
tumour cells.
Specific active immunotherapy by the injection
o f tumour cell Vaccines' was tried early in last
century but was given up as unprofitable. The
method has been modi fied recently by usi ng pirificd
tumour cell membrane antigens and tumour cells
treated with neuraminidase to increase their
immunogenic potential.
Nonspecific active immunotherapy employs
BCG and nonliving C atyntbactctium parvLim,
M at he, the leading proponent o f cancer
immunotherapy has reported very good results in
acute leukeni ia, fnllowi ng combi oed treatment with
BCG and allogeneic or autochthonous leukemia
blast cells. Intralcsional B C G in m alignant
melanoma has been reported to induce complete
remission in a high percentage ofparicntS- It has
also been used against intradermal recurrence o f
breast cancer following m astectom y.
D im troth Lo robe nxene bat been tried in the
treatment of squamous and basal cell carcinoma of
the skin. Clucan, a pyran copolymer derived from
m icroorganism s, and le van ii sole, originally
introduced as an anthelmintic, have been tried for
stim ulating cell m ediated im munity and
macrophage functions. Interferons have been
employed in the treatment of leukemias.
Specific adoptive immunotherapy has been
attempted with lymphocytes, transfer factor and
"immune RNA .The donors have been persons who
have been cured o-f their neoplasms or specifically
immunised against the patient's tumour.
Lymphokinc activated killer (LAKJ cells obtained
by treatm ent o f the natural killer cells with
interleukin-2 have been found useful m the treatment
of certain malignancies, such as renal carcinomas.
Immunotherapy is ineffective in the presence
of a large mass of tumour cells. Its role appears to
be more in geinng rid of the residual maiignant
cells after the gross tumour has been removed. The
best results in the treatment of cancer apparently
follow an integrated approach to therapy,employing
surgery, radiotherapy, chem otherapy and
immunotherapy.
C o p y rig h te d m aterial
 m
■3d

sF-*
jZ

C o p y rig h te d m aterial
 Im mu nohematology

Blood has held a mysterious fascination tor llh tro:n blood transfusions or mothers o f infants with
the Jaiwn of time. It was considered the essence of hemolytic disease. The main blood group system;
life and was believed to cure diverse diseases and with the dates of their discovery are shown below.
restore youth and vitality to the aged. Blood
transfusion has been attempted from very early dines ABO 1900 Duffy 1950
bur such attempts were fruitless and often fraught MN 1926 Kidd' 1951
with disastrous consequences. Blood transfusion P 1926 Diego 1955
became sc ientitically fea^ ible only after the discovery Rh 1940 Yt 1956
of blood groups by Landsteiner. Lutheran 1945 Kg 1962
In bis original experiment, Landsteiner (1900) Lewis 194b Dombrock 1965
cross tested serum from himself and five of his Kell 194b Colron 1967
colleagues against their red blood cells. Three
distinct patterns o f agglurination were observed. Some antigens have been identified that occur
Cells which foiled to agglutinate with any of the very rarely, being limited to certain individuals or
serum samples were des ignated group O, while cells families. These have been termed 'private antigen s’.
agglutinating in the two different patterns were
called groups A and B. respectively. T h e fourth ABO BLOOD GROUP SYSTEM
group A B was described Inter bv his pupils von The A BO system contains four blood groups and
Decastallo and Sturli (1902). In 1930, Landsteiner is determined by the presence or absence o f two
was awarded the Nobel Prixe for his discovery of distinct antigens, A and B, on the surface o f
Human blood groups. erythrocytes. Red cells of group A cany antigen A,
The ABO system is the most important o f all cells o f group B antigen B and cells of group AB
the blood group systems and its discovery made have both A and B antigens, while group O cells
blood transfusion possible. No other blood group have neither A nor B antigen. The four groups are
antigens were discovered for the next 25 years. Using also distinguished by the presence or absence o f
rabbit antisera to different samples of human red two distinct isoantibodies in the serum.The serum
cells, Landscr ii ier and Levi ne (1926) discovered the contains the isoantibodies specific for the antigen
M N and P antigens. Landsteiner and Wiener (1940) that is absent on the red cclL The serum o f Agroup
rinsed rabbit and guinea pig antisera against Rhesus A individual has anti-B antibody,group B has ami-
monkey erythrocytes and tested them against human A and group O both anti-A and anti-B, while in
red cells. This led to the discovery of the "Rhesus group AB both ami-A and anti-B are absent (Table
(Rh.) factori, Many more blood group antigens have 21. 1).
been identified subsequently, mostly by studying G roup A is subdivided into A l and A2-
antibodies in patients who had received multiple Antiserum of group A agglutinates group A l cells

C o p y rig h te d m aterial
powerfully hut A 2 cells only weekly. A bo ut HO per
rent of group A blood is A t and 20 per cent A 3 -
T h e subgroup*; o f A antigen art represented in group
A R al&u. T h e recognition o f group A subgroups
increases the number o f A B O pherrotypes from four
to sU : A l h A 2 h A 2 B , A 1 B and O , Other A
subgroups (A 3 , A 4 j A 5 ) have also been described
but they arc not clinically rdevant-
Blood group antigens are inherited according
to simple Men deli an laws. T heir synthesis is
determined by allelomorphic genes A, b and O.
Oenea A and B give rise to the corresponding
antigens, hut O is an amurph and does not product:
any antigen- The frequency of ABO distribution
differs in different peoples, Group O is the most
common group and AR the rarest. The ABO
distribution in Britain is approximately 0 -4 7 per
cent, A - 42 per cent, R - B per cent and AH —■3
per rent- In India, the distribution is approximately
0 - 4 0 per centf A - 22 per cenrf li - 33 per cent and
AB —5 per cent,
Anri-A and anti-B isoantibodies appear in the
serum of infants by about the age of six months
and persist thereafter. These are called ‘naturalt
antibodies because they seem to arise under genetic
control without any apparent antigenic stimulation.
However, it n likely that they develop as a result of
unidentified environmental stimuli with the blood
group-like antigens present in bacteria or other
sources. Natural anti-A and anti-B antibodies are
IgM saline agglutinating antibodies reacting
optimally between 4 and IS ‘’C but less active at
37 'A-, Immu nc isoantibodics may develop following
A H O incompatible pregnancy'or transfusion. M ore
A A B, 0
B B A, 0
AD A and ft A ,B ,Q
0 N ont None
com m only they result from the injection o f
substances containing blond group-like antigens,
such as horse serum or bacterial vaccines made
from media containing horse or hog extracts.
Immure isoantlbodics are 'albumin agglutinating'
IgG antibodies reacting optimally at 37 °C and
acting as hemolysins in the presence of complement.
They are clinically more important than natural
IgM antibodies and may cause m ore sever*
transfusion reactions.
Red cells of all ABO groups possess
a common antigen, the H antigen or H substance
which is a precursor for the formation of A and R
antigens. The amount of the I I antigen is related
to the A B O group of the cell, group O cells having
the most and AH the least amount. D u e to its
universal distribution, the H antigen is not
ordinarily im portant in grouping or blood
transfusion. Bhcnde ct al (1952) from Bombay
reported the very rare instance m which A and B
antigens as; well as the H antigens are absent from
red cells. This is known as ‘Bombay1or OH blood.
Such individuals have onti-Ah anti-B and onti-H
antibodies and their sera are incompatible with all
red cells except of those with the same rare blood
gnmp.
A t B and H antigens are glycoproteins. They
are not confined to erythrocytes but can be detected
in almost all tissues and fluids of the body. While
these antigens are always present in tissues, they'
are found in secretions (saliva, gastric juice, sweat)
of only about 75 per cent of all persons. Such
persons arc called 'sccretcrV and those who lack
blood group antigens in secretions are called
anti-B B, AB
anti-A A, A B
None None
A otL-A ind ifln-ft A, B. A B
C o p y rig h te d m aterial
lnon£ecretors\ The secretion af" ARH antigens in
controlled by two allelic genes be a ltd sc. J ndividua Is
who arc homozygous or heterozygous b)r St are
se cre to ry while rhose who ire se-se
AdiUHictdn.
A and El antigens are ulso found in certain
animals and plants. They hive been extracted and
purified commercial!/ from the stomach of horses
and hogs. Blood group anribodict are also found in
some animals. Substances spedficilly agglutinating
A or R antigens have been detected in Some plants.
A potent anti A 1 agglutinin has been extracted fiotn
DoUchtK hiflarua and ant] H from Viex ctUOfMCUf-
Blood group agglutinins ol plant origin are known
as icftinh'.
hi- L r.V
Levine and Stetson [1939) demonstrated a new type
of antibody in the serum, of a woman who devdojied
severe reactions following transfusion of her
husband's ABO compatible blood. She had recently
delivered a stillborn ml in I with hemolytic disease.
They suggested that the woman may have been
sensitised by some antigen inherited by [1111 fetus
from its lather. Lindsteinei anil Wiener (194i))
identified in the red celh ol the miioii tv of persons
tested, an an rigen that rcac ted wi th rabbi r an tisr-ni ni
to Rhesus monkey erythrocytes, This antigen was
culled the "Rhesus' ut Rh factor. The "new Lvpe' oi
antibody described by Levine and Stetson was
identified as the anri-Rh factor antibody. Wiener
and Ftecers (1940] demonstrated and-Kh antibody
in same peisons who hid received A BO compatible
transfusion. Levine and colleagues [1941) proved
that Rh sensitisation wns the cause o f hemolytic
disease o f the newborn.
The R h system is complex and its study is
complicated by the existence o f two different
theories and nomenclatures for the genes and
antigens. Wiener proposed that Rh antigens are
detc rm ined by any one of several ,ill die genes which
may appear at a single locus nnd govern the
production of the appropriate agglutinogen on the
Surface- ot erythrocytes. Each agglutinogen is in turn
made up of one or man; antigens. Fisher, on the
other bund, postulated that Rh antigens are
d L-ca rm ilie d by three p a irs o f clo s e ly lin k e d
allelomorphic gene a, C c, D d and Ee, Every
individual possesses one member of each pair o f
iheye gent^ derived from each parent, Each gene
would be responsible for the production of a specific
antigen* which l o u hi lie detected Eiy its Specific
antibody,
The designations employed by the cwo systems
Mr the difterenr Rh types are as follows:
Fisher H5tner
Rh positive CDe Rhl
cDE Hh2
l'D c; Kbo
CDE Rhz
Rh negative Cdc rh/
cdE rh//
ode rh
CdE thy
Kir routine purposes-, the typing of persons as
Rh positive or negative depends on the presence or
absence of antigen D (klio) on red cells and hence
car be accompli*hed hy testing with anti D {anti-
Rh) serum. This is because D is the most powerful
RH antigen and accounts tor rhe vast majority of
Rh inconipaabihiy reactions. The distribution of
Rh positives J itiers- in difterenr races. Among people
o f European descent, about S5 per cent ate Rh
positive and 15 per cent negative. Among Indians,
approximately 93 per cent arc Rh positive and 7
per cent negative.
A variant of IJ is known as DlL, Red ceils of Du
subtype react with some but not all inti-D sera.
Though Du cells may nor be agglutinated by anti-
1> sera, they absorb the antibody on their surface.
The Du subtype can therefore be detected by
reacting red cell with anti-D serum and then doing
. l direct Coombs test- For the purpose o f blood
donation, Du cells arc considered Rh positive. Bur
when a Du individual requires transfusion, it is
advisable to -.i-.c Rh negative blood because be is
C o p y rig h te d m aterial
capable olf'being immunised by standard Rh positive
blond.
I’htre arc no natural anti-Rh antibodies in the
serum.. T hey arise only as a result o f Rh
incompatible pregnancy or trunsfimon.
The Lewis blood group system consists of two
antigens LeJ and Le^, Ir differs from other blood
group systems in that the antigens are pre&ent
primarily in. the plasma and saliva. Red cells acquire
the antigen by adsorbing them from plasma. The
Lewis phenotypes are closely related to the ABO
group and to the secreEor status o f an individual.
M altma Hy oecum ng f.ewis jnribijdiies are frequently
found in the sera o f p e rso n s la c k in g the
corresponding antigen.
In the MN system, usmg ratslsir: antisera, persons
were originally classified into three groups— M ,
N and VIN. An antigen, S„ was later added to this
system.This system has expanded to include at least
28 antigens.
Blond group systems other than ABO and Rh
arc of little clinical importance as they do not usually
cause transfusion reactions or hemolytic disease.
They have applications in genetics* anthropology,
tissue typing and torensic medicine. As blood group
antigens are inherited from the parents, they are
often useful in settling cases of disputed paternity;
T h e existence o f several different blood group
a n tig e n s m a k es it a lm o st im possible to obtain,
perfectly m atched blood for transfusion. B u t in
routine transfusion practice, only the A B O and R li
antigens are relevant. 1 he other antigens are COO
w eak ro he of im p o rta n c e . S a fe ty ill b lo o d
transfusion requires that the following conditions
be satisfied in choosing a donor.
1. T h e recipient's plasma should not contain any
a n rlb o d y th at w ill d am ag e th e d o n o r's
erythrocytes.
2. The donor plasma should not have any antibody
that will damage the recipient's red cells.
.1. The donor tftdcelk should not have any antigen,
that is lacking in the recipient. Jl the transfused
Cells possess a "foreign antigen', it will stimulate
an immune response in the recipient.
Ideally, the donor and recipient should belong
to the same A B O group. It used to be belt] that Q
group cells could be transfused to recipients of any
group as they possessed neither A nor B antigen.
Hence the O group was designated as the- 'universal
donor', The anti-A and anti-B antibodies in the
transfused O blood group do not ordinarily cause
any damage to the red cells of the A or 3! group
recipients because they will he rendered inelfective
bv dilution in, the recipient's plasma. But Mime O
group plasma may contain isoanribodics in high
dtres (1:20U or above) so chat damage to recipient
cells may result- This is known as the 'dangerous
O group'. Tlic ant]-A antibody in die O group blood
is generally more potent than, the anti B antibody.
Hence the O blood group is more likely to cause
an adverse reaction when given to the A group
recipients than to those o f die B group. Wlnle the
O group blood with low tltTe antibodies may be
transfosed to a patient of any other group in dire
emergency, this practice should never be employed
as a routine, Transfusion of large quantities of the
O group blond to persons ol any other group may
cause adverse reactions.
Due to the absence of iroantibodies In plasma,
the AR group perrons were designated universal
recipients1. AB group donors may not always be
available due to their rarity and ir may, on occasion,
he necessary to use- demurs of othe r groups, In Such
cases, group A blood is safer than group B, because
the ami-A antibody is usually more potent chan
the anti-B antibody,
Rh compatibility is importanr only when the
recipient is Rh negative. An Rh positive person
may safely receive cither Rh positive or negative
blood. But un Rh negative individual receiving Rh
jjusitive blood may form antibodies against the Rh
C o p y rig h te d m aterial
IBS A Te*rt>3IJK . 01
anfil'en. A subsequent transfe-i on with Rh po^-Hive
blood may then cause an adverse reaction. An
additional r i s k i n w o m c r ■> I i l i sensitisation I c . L U m g
td hemolytic disease o f the newborn. Therefore it
is particularly important that Rh negative women
who arc not past childbearing age receive only Rh
negative Moth!
Besides A BO grouping and Rh typing of the
donor and recipient, it is invari.ihly necessary hefiire
transfusion to perform a 'cross matching^ to ensure
that the donor's blood is compatible with the
recipient' blood. The rouune procedure used in
most blood bank* is a rapid cross match by the tile
or slide method, This is done in two parts - the
major cross match where the donor red cells are
tested against the recipients scrum. and the minor
cross match where the recipient's cells are tested
against the donor scrum. One drop o f a v'ti
suspension of donor red cells in saline is added to a
drop of the recipients scrum on a porcelain tile or
a glass slide, mixed and observed for agglutination.
Though in most cases agglutination occurs early,
it may sometimes be delayed. 'lire result is to be
read, m icroscopically and under low power
micTuscope, after incubation in a moi-t chamber
for 1 0 - l s mi notes at room temperature. In the
minor cross match, the same is repeated using
rcci |'Lent cells and donor Rcrum. Orly the m.ijor
cross match is done ordinarily.
The saline slide test does not detect Rh and
other minor incom patibilities. The most
discriminating method is the Coombs cross match
where washed donor cells and recipient serum are
incubated in a water bath ar 37 *C for two hours
and a direct Coombs test done. Thii detects all
incompatibilities^ rndmiing incomplete anribotues.
Following an incompatible blood transfusing
the red cells may undergo clum ping and
intravascular hemolysis or they may he coated hy
anrihodics, engulfed by phagocytes, removed from
circulation and sut eeted to cxtravascular lysis,
Incompatible transfusion may be accompanied by
symptoms such as sluveriug, Singling sensation.
Mir.rubiology ►
excruciating headache, constricting precordial
discomfort and severe lumbar pain. Hypotension*
cold clammy skin, cyanosis, feeble pulse and other
signs of collapse may he seen. Jaundice, hematuru*
oliguria and anuria may follow.
Some transfusion reactions may be due to
immunological processes other than blood group
incom patibility. Rigor, u rticaria and other
mandeslal inns often occur due to the recipient being
bypenen* it ivc to some allergen present in the donor
blood. Serious reactions follow when hemolysed
or contaminated blood is transfused.
Whenever any reaction occurs, the transfusion
should he stopped immediately. The remainder of
the donor blood -should be sent to the blood bank
for investigation.
The most common complications following
blood transfusion are o f infectious origin.
Transfusion of blood contaminated by bacteria may
Lead to endotoxin shock or septicemia. Gross
contamination can be recognised in most cases by
inspection of the blood before transfusion, as
hemolysis is usually apparent. Such contamination
can be eliminated by proper techniques of blood
collection and storage,
The most important infections transmitted at
present by blood transfusion are the H IV and
hepstriis viruses- Several cases of transfusion-
induced AIDS have occurred before HIV screening
of donors became mandatory. However, screening
may not detect H lV infected donors during the
window period when they are infectious. Hepatitis
B , C, D and possibly others can be transmitted by
transfusion. Screening for the hepatitis B surface
antigen can exclude most H B V carriers but the
avaLiable serological tests against other hepatitis
viruses axe not quite satisfactory.
Despite diligent S c r e e n in g , there exists a small
risk (about 1 in 3 0 0 ,0 0 0 ) risk o f transfusion
associated HIV, H BV and H C V infections. The
variant CJD prion is another risk in endemic areas
like the UK where it is mandatory tn screen donors
for the prion and to remove leucocytes from blood
C o p y rig h te d m aterial
before Transfusion, a* a precautionary measure.
Cvto megal-Dvirus transmitted hv Iran?-fusion may
cause an infectious mononucleosis-like syndrome.
Syphilis may be transmitted by transfusion of fresh
blood from an infectious donor but not if the blood
has beer stored for three days or more before
transfos ion, Mrdaria is another disease transm iss iblc
by transfusion.
W h o le blood tran sfu sio n is being replaced
increasingly by blood component therapy, w hich
causes lewer complications and results in more
opti m id uti Iisji fion of hu man blood vi+i Leh is a scarce
commodity. For example, in anemia, packed red cell
transfusion is more beneficial titan whole blood as
it provides greater oxygen carrying capacity' with
less circulatory overload and minimal clecrrolyte
disturbance. Frozen red Cells ate available for
transfusion in patients with rare hlood groups.
Similarly, leucocyte and platelet concentrates are
available for Hjteclfic needs. Plasm a cryoprecipitatcH
and Factor V III are routinely used in Treating
hemophilia. O ther coagulation factors arc also
available for different coagulation disorders. Such
plasma products are manufactured from pooled
human blood and may transmit HI V , hepatitis type
11 or C virus and other infectious agents unless great
care is taken in the selection of donors and in
manufacturing processes, An illustration o f the
enormity of the risk was the tragedy in France in
the inid."l9S0sn when tainted blood caused I UV
infection in over 4000 persons, of whom hundreds
died of AI US in the next few yearn. Charges of
criminal negligence were framed against the persons
considered responsible, front the Frame Minister
downwards. Though the Prime M inister was
acquired. the Health Minister, the Head of the
National Blood Transfusion Service and many other
top officials were coovicted-
Alniosl all adverse reactions o f transfusion car
be eliminated by autologous blood transfusion
which in rapidly becoming po|iular, Here, blood is
collected from the individual himself and stored
for use during elective surgery. Blood collection
may be started a month before the expected date of
transfusion. Autologous tram* us ion elim inates nut
only infectious complications but also those due to
minor blood group incom patibilities and
hypersensitivity.
When an Rh negative woman carries an Rh positive
fetus, she may be sensitized against the Rh antigen
by the passage of tetal red cells into the usaLcrriai
circulation. Minor transplacental leaks may occur
any time during pregnancy but it is during delivery
that fetal cell-s enter the maternal circulation in large
numbers. The mother is usually sensitized only at
the first delivery and, consequently, the first child
escapes damage (except where the Woman has been
sensitised already by prior Rli incom patible
transfusion). During a subsequent pregnancy. Rh
antibodies of the IgG daw pass; from the mother
to the fetus and damage its erythrocytes. This is
the pathogenesis of the hemolytic disease of the
newborn. The c li nleal features may vary from a mere
accentuation of the physiological jaundice in the
newborn to erythroblastosis fetalis or intrauterine
death due to hydrops fetalis.
H em olytic disease docs not affect all the
offspring o f Rh incom patible marriages. Its
incidence is much less than the expected figures.
The following facror^ influence rhe incidence o f
hemolytic disease due ro Rh incompatibility:
Not every Rh negative individual
form s Rh antibodies following antigenic
stimulation. Some fad ro do so even utter repeated
injection of Rh positive cells. They ire called
nnnrefipondera. The reason for this inimunologkaJ
unnesponsiveness is not known.
Rh
Immunisation is more Likely to result when the
mother and focus possess rhe same A BG group-
W heo Rh and A liO incompatibility coexist, Rh
sensitisation m the mother is rare. In this situation
C o p y rig h te d m aterial
the fctil cells entering the maternal circulation art:
believc-fi to be destroyed rapidly bv the A R O
antibodies be tore they can induce R h unt iliodi es.
The flir^t child
usually escapes disease because sensitisation dcuis
only during its delivery. The risk to the infant
increases with rath successive pregnancy
A n in d iv id u a l m ay
be liomozygous or henerozygous with respect
to [} antigen. W h e n the lather in homozygous
all his children will be Rh positive, W h e n he
is hetero zygo us, h a lf his ch ild re n w ill be K h
positive.
("i E T T J1 ♦’ »M T f t [a
M o lt Rb antibodies are o f the igt I class, and being
'incomplete antibodies', they do nut agglutinate Rh
positive cells in satine. A minority are complete
(saline agglurinating) antibodies of the IgM class.
These are not relevant in the pathogenesis o f
hemolytic disease as they do not traverse the
placenta.
lg G a n t i-D antibodies m ay be deterred by the
followiog teehni^ucy: ( I) uidng a colloid m edium
such as 20 pc r cent bovine sc rum album in ■ (2) usi ng
red cells treated w ith enzym es surh as trypsin,
pepsin, fip n or h ro m d in ; and (.1) by the indirect
C o o m b s test. T h e last is the most sensitive method.
Rh typing should form a part of routine antenatal
examination. W h e n the woman is Rh negative, and
her husband Rh positive, fetal co m p ila tio n s should
be e x p e cte d . W o m e n w ith R h in c o m p a tib le
pregnancies should be screened for Kh antibodies
hy the indirect C o o m b s tesi at 3 2 -3 4 weeks o f
pregnancy and at m onthly intervals thereafter. T h e
appearance of Rh flutilntdies during ynegnanrv or
their increase in title, if they were present already,
w o u ld prove th at the fetus is R h p o sitiv e . ] f
am niocentesis is indicated, dem onstration o f R h
antigen in the am niotic fluid w ould also prove that
the fetus is Rh positive. In the ease of hemolytic
disease o f the newborn, the maternal serum will
show Rh antibodies in the indirect C-oombs test
and the infant's ret] Cells will give a positive m the
direct Coombs test.
W h en hem olytic disease is diagnosed
antepartum, an intrauterine transfusion with Rh
negative blood max be indicated Red cells
introduced into the fetal peritoneal cavity will find
their way into the circulation and will survive
normul]y. Rremature delivery followed by
transfusion may he necessity in some cases. When
a baby is born with hemolytic disease, exchange
transfusion with Rh negative A BO compatible
blood is the treatment of choice.
Remarkable success has been achieved in the
prevention of Rh isoim m u im ation by the
administration of anti-Rh lgG antibody at the time
when the antigenic stimulation is expected to take
place. The passively administered antibody may
prevent isoimmunisarion by a negative feedback
mechanism or hi afferent inhibition. The
recommended practice is to inject 1 0 0 -3 0 0 Jig of
Rh immune (gG to an Rh negative woman
immediately after delivery. To he effective, this
should be employed from the first delivery unwards.
The Rh immune globulin for the purpose is
prepared from human volunteers.
ABO S t a e r n t f i K ; ($<*:**.*■*
Maiernofetil A BO incompatibility is very common
and in a proportion of these, hemolvtic disease
occurs in the newborn. In persons of blood group
A or B, natural antibodies arc IgM in nature and
so do not cross the placenta to harm the fetus.
However, in persons of blood group O . the
isoantibodics are predominantly lgG in nature.
Hence ABO hemolytic disease is seen largely in O
group mothers, bearing A or B group fetus. As
A HO hemolytic disease is due to naturally occurring
maternal isoanribodics, it may occur even in the
C o p y rig h te d m aterial
fir s t b o r n , w it ho lit prior immunisation. ABO
hemolytic disease is much m ilder than RK disease,
probably because erythrocytes o f the newborn haft
(ewef A Or B antigenic sites as co m p ile d tfl adult
erytlirocyTrs- line direct Ctwmh-^ Test is therefore
often negative in this condition, while the indirect
Coombs test (neonatal serntn with type ■ s pcc i fic
adult erythrocytes) is more commonly positive.
Peripheral blood smear characteristic illy shows
spherocytosis.
It has been shown that some diseases may influence
blood group antigens. Blood group antigens have
been inputted to become weak ill leukemia. The
reason for this is nor known. I’bc acquisition of H
antigens by Group A persons has been observed
following some infections. The antigen is believed
A n & rtu n K ( ' and P M ed* 1994. &-j>ntjfic Bi&rtofTramftfiijrpn M n lin iM . Philadelphia: &JUmdeffc
Sl!ltjli'.t ( JB cl aL. 19%. Th* rink of tiaii.*fiision-traiuanLHcd vuaL lEdu.TMn--. j\'eiy B nglJ Med33+1645,
to come from the infecting microorganism.
Red cell suspensions contaminated with certain
bacteria, such ns TVeudomonfl* aeruginosa,
become ngglutillable by all blood group :-cra and
even hv normal human sera. This phenomenon,
known as the Thom sen—f^mdcnreich phenomenon,
is due to the unmasking of a hidden antigen normally
present o r all human erythrocytes. This is called
the T antigen. Anti-1 agglutinins are normally
present in human sera. Such panagglutii Lability of
red cells has occasionally been observed in persons
suffering from systemic bacterial infections.
Several investigators have attempted to correlate
blood group and susceptibility to certain diseases,
It has been shown that duodenal dicer is mote
frequent in persons of blood group O than in others.
An association has also been established between
group A and cancer of the stomach.
C o p y rig h te d m aterial
 Staphylococcus
Staphylococci arc Gram positive cocci that occur
in grape-like clusters. They are ubiquitous and form
the niio^t common cause of localised suppurative
lesions in human beings. Their ability ro develop
resistance to penicillin and Other antibiotics
enhances their importance as a human pathogen,
especially in the hospital environment.
Staphylococci were brat observed in human
pyogenic lesions by von Recklinghausen in 1871.
Pasteur (18S0 ) obtained liquid cultures ot the cull!
from pus and produced abscesses by inoculating
them into rabbits. It was Sir Alexander Ogstun, a
Scottish surgeon, who established conclusively the
causative role o! the coccus in abscesses and other
suppurative legions (IPSO). He also gave it the name
Staphylococcus (Staphyle, in Greek, meaning
‘bunch of grapes': kokkos, meaning a berry) dtic to
the typical occurrence o f the cocci in grape-like
dusters in pus and in cultures. Ogstorl noticed [bat
nonvindent staphylococci were also often present
on skin surfaces.
M ost staphylococcal strains from pyogenic
lesions were found to produce golden yellow
CoLotticJ, And the strains from normal skin, white
colonics on solid media. Rosenbach (1884) named
them Staph, aureus and Staph. albus respectively.
Passet (lkS5) described a third variety, Staph.
vrVreiw, producing lemon yellow colonics. However,
the association between virulence and pigment
production was not found to be constant.
Many other properties were proposed as
indicators of virulence. These included hemolysis,
gelatin liquefaction, lipolytic activity' production of
urease, phosphatase and others, but none of these
was found reliable. The most constant association
was between virulence and production of the enzyme
coagulase, and to a lesser extent fermentation of
mannitol. Staphylococci were therefore classified
into two groups: Staph, aum is (formerly also called
Srapft pyog^tTl containing strains giving a positive
L-ougulasc test, fermenting mannitol and usually
being pathogenic, and Staph- ^fdcrznrdu { formerly'
also called Staph. a J k s ) containing coagulase
negative, mannitol nonfetmenting and usually
nonputhogeuic strains. Tlie genus Staphylococcus
is now classified into 32 species and IS subspecies
based ot] the chemical composition o f their cell
wall components and other properties. Besides
Staph, dum u, two euagulase negative species -
Staph. cpriJcrmith*' St*phr haemo/ytrcti? and Sfcph.
sapraphytiais- can also cause human disease. Some
other coagulase negative species such as Staph,
h o m in k and Staph, eapffus are part o f the
commensal flora of the human skin. Other species
□re para-dhc on animals.
£L I M S
They are spherical co cci,
approxim ately 1 pm in diam eter, arranged
characteristically in grape-likc clusters (Fig. 22.1).
Cluster formation is due to cell division occurring
in three planes, with daughter cells tending to
remain in close proximity, They may also be found
singly, in pairs and in shori chains of three or four
tells, especially when examined from liquid culture.
Long chains never occur They are nonmotile and
nonsparing. A few Strains possess microscopically
visible capsules, particularly in young cultures.
Many apparently noncapsulated strains have small
amounts o ( capsular material o r the surface. They
C o p y rig h te d m aterial
Stain readilv with aniline dvc! -and are uni hum Ip
G n im positive. U n d er the influence o f p en icillin
and certain chem icals, they m ay change do L form s.
........... They grow readily
on ordinary media, w ithin a tetUpCntm t range ot
10 -4 2 PC, the optimum heirg 37 and pH 7.4—
7.6. They me aerobes and facultative anaerobes.
On nutrient ag-.u, after incubation tor 24 hours,
the Colonies arc large {2 - 4 mm diameter), circular,
convey, sm ooth , shiny, opaque and easily
cmulsitiablc. M m t strains produce golden yellow
pigment, though some (nay be white, orange or
yellow, The pigment does not diffuse into the
medium. Pigment production occurs optimally at
22 CIS and only in aerobic cultures. Pigment
production l-s enhanced when 1% glycerol
mornsacetate or m ilk is i nmrpnra ted i n the mrdiu m .
The pigment is believed to be a lipoprotein allied
to carotene.
On nutrient agar slope, iht confluent growth
1. ' • -
prese nth a clu a t t t r i slic ' o il- p.i in i1 appearance. T h e
colonies o r blond agar are sim ila r to those on
nutrient agar. Most strains arc hemolytic, especially auwti but not by other species. They are catalase
when incubated under 20-25% carbon dioxide. positive (unlike streptococci) and usually hydrolyse
Hemolysis is marked on rabbit or sheep blood and urea, reduce nitrates, to nitrites, liquefy gel atin Jnd
weak on horse blood agar. are MR and VP positive but indole negative. Most
T h e y grow on M a c C o n k e y ’s m e d iu m , strains are lipolytic and when grown on media
producing smaller cola rues that art1 pink due Co containing egg yolk, produce a dense opacity,
lactose fermentation. Production ot phosphatase can hr demonstrated by
In liquid media, uniform turbidity is produced. culturing o r nutrient agar containing
Several selective media have been devised lor phenolphthalein diphosphate. When such a culture
isolating Staph, m rtvi from s]H.Lcimcn5 such as feces is exposed to ammonia vapour, colonies assume a
containing other harteria. 1 he sc include m cilia bright pink colour due to the presence of free
containing 8--10 per cent N a d (salt-milk agar, a-a.Ec phcnolphrhaleim This is a useful screening
broth), lithium chloride and tellurite (Ludlam's procedure fnr differentiating Staph. aureus from
medium), ami polvrnvxin, For primary isolation, Staph, epidermidit in mixed cultures, as rhe former
sheep blood agar is recommended. Human hlooil gives prompt phosphatase reaction, while the latter
should not be used as it may contain antibodies or is usually negative Or only weakly positive.
other inhibitors. Staph- aareut strains usu-dlv exhibit the lolliKwiug
r : T h e y fe rm e n t a cha raetcri sties: {!) coagulant positive; (3) greater
number of sugars, producing acid bur no gas. Sugar biochemical activity, ferment mannitc; {3 ) produce
fermentation is o f no diagnostic value except for cle a r h em o lysis on blood agar; (4) p ro d u ce a
m annito l, whieh is usually fermented bv Staph. golden yellow pigment; (d) liquefy gelatin;

C o p y rig h te d m aterial
194 ’i Textbook ot Microbiology ►
p ro d u ce p h o sp h a ta s e ; (7) in a m e d iu m
containing potassium : i.]l u r LI c . reduce tellurite to
form black colonic?; and (8) produce thermostable
nuclease!; w hich can be dei:v mstiaced by die ability
of boiled culture? to degrade DNA in an agar
diffusion test.
R e a u ta n c e Staphylococci are among the more
resi-tant of nemsporing bacteri.L. Dried on threads,
they retain their^vjability' for H months- They
have been isolated from dried pus after 2 - 3 months.
They may withstand 64) °C for 30 iriiilLiKS. Their
thermal death point in f,2 °C lor 30 minutes. Some
staphylococci require 80 DC for one hour to be
lulled. Heat resistant strains have the ability to
grow at ahiglicT temperature, even aE 4 ? C. Most
strains grow in the presence of 10% N aG and some
even in N a d , T hese features are o f
si.gnificance in food preservation.
Theyrt>^t l<Hi phenol for 15 minutes. Mercury
perchloride 1 kb solution kills them in 10 minutes.
Many aniline dyes arc strongly bactericidal* crystal
violet being lethal at a concentration of one in live
hundred thousand and brilliant green, one in ten
million.
Fatty acids inhihir the growth of staphylococci,
the highly unsaturated acids having a more
powerful acrior. on coagulase positive than on
coagulasc negative strains. Staphylococci arc
uniformly resistant to Lysozyme but some
micrococci arc sensitive to it. StaphyLococu are
generally sen:-ltive to Ivsnstaphiii— p mixture of
enzymes produced by a particular strain of Staph,
epidern iidi ■■.
Staphylococci were uniformly sensitive to
penicillin originally, th-.mgfi ticcasional strain- from
the pncantibiotic era have been found retrospectively
to be capable of producing penicillinase. Soon after
penic illin came to be used c 11me illy, resistant strs ms
began to emerge, first in hospitals and rhen in the
community at large.
Penicillin resistance is ol three types
1. production of beta Lactamase (penicillinase)
which inactivatcs pcniiciIi'.n by splitting the beta
lactam ring. Staphylococci produce four types
of penicillinases, A to D. 1 Jospital strains usually
form type A penicillinase. Penicillinase :■* an
inducible enzyme and its production is usually
controlled by plasmids which are Transmitted
hy transduction or conjugation. The same
plasmid may cany genes for resistance to a range
of other antibiotics and heavy metals.
2- Changes in bacteri.iL surface receptors, reducing
binding of bcta-lactam antibiotics to cells. TV'S
change is normally chromosomal in nature and
is expressed more at 30 UC than at 37 ^C. Tins
resistance also extends to cover beta lactamase
resistant penicillins such as methicillin and
cloxacidins. Some of these strains may show
resistance to Other antibiotics and heavy metals
also and cause outbreaks of hospital infection,
These strains have been called 'epidem ic
methicillin resistant Staphylococcus aureus' or
EM R SA (as methicillin is an unstable drug,
cloxacillin is used fir sensitivity testing instead).
3. Development of tolerance to penicillin, by which
the bacterium is only inhibited but not hilled.
Staphylococci also exhibit pi aem id-borne
resistance to erythrom ycins, tetracyclines,,
aminoglycoside? and almost all clinically useful
antibiotics except vancomycin.
P a lh o g c n icily an d v iru le n c e ;
Staphylococci produce two types o f diseases—
infections and intoxications. In the former the cocci
gain access to damaged ski n, mucosal or tissue sites,
colomi.se by adhering to cells or extracellular matrix,
evade host defence mechanisms, multiply and cause
tissue damage. In intoxications, the disease is caused,
by the bacteria] toxins produced cither in the
infected host or preformed in vitro. A number of
staphylococcal factors, both cell associated and
extracellular, have been identified, which may
influence virulence. However, apart fr o m the
exrnnxins wliiich cause specific clinical syndromes,
no other factor has a decisive role in pathogenesis.
The virulence factors described include the
follow Lug;
C o p y rig h te d m aterial

1- The cell wall polysaccharide peptidoglycan
confers rigidity and structural integrity to the
bade rial cell. It activates complement and
induces release of inflammatory cytokines,
Z Tcichoic acid, an antigenic component of the
cell wall facilitates adhesion of the coed to the
host cell surface and protects them from
complement-mediated opsonisation.
’I . C a p su lar polysaccharide surrounding the □ ell
wall inhibits opsonisation.
1, Protein A prese nt on most Staph aureus strains
has m a n y hiulogical p ro p ertie s, including
chem alR ctic, antiphagocytic and an ti-
complcmentary effects. It also induces platelet
damage and hypersensitivity. Protein A binds
to the Pc terminal of IgG molecules (except
IgG J), leaving the Fab region free to combine
w ith its specific antigen. Protein A-bearing
staphylococci coated with any JgG antiserum
will be agglutinated if mixed with its
corresponding antigen. T h is procedure known
as L'oaggluti nation has m any apI ications, as for
streptococcal grouping and gonococcal typing-
Piotein A is a B cell mitogen. It has also been
used as a ligand for isolation o flgG .
Z Clumping factor, another surface protein is. the
“bound w agulasc1*which is responsible for the
‘slide coagulasc' test. When a saline suspension
of Staph, jurats is mixed on a slide with a drop
of human plasma the cocci arc clumped. Ilie
slide coagulasc test is routinely used for the
identification o f Sraph. aureus isolates.
Capsulatcd strains may sometimes show a
negative test because the dumping factor may
be enveloped by the capsular polysaccharide,
1. Coagulasc is an enzyme which hrings about
clotting nf human or rabbit plasma. It acts along
with a 'coagulasc reacting factor’ (C R F) present
in plasm a, binding to prothrom bin and
converting fibri nogen to fihrin. Coagulasc docs
not clot plasma of guinea pigs and some other
species because they lack CRF- Calcium or other
dotting factors are not required for coagulasc
action. Eight types o f coagulasc have been
identified. Most human strains iomi coagulase
type A, Coagulase and clumping factor (the so
called 'boundcoagulase1) differ in many respects.
Coagulase is an enzyme secreted into the
medium. It requires rhe cooperation o f C R F for
its action. Bound cougufesc ™ 3 heat stable
constituent of the cell surface and its action is
independent of CR.F. Only one type of clumping
factor has been identified. Staph, aureus strains
usually form both coagulasc and clumping
factor. Coagulasc test is the standard criterion
for the identification of 5t^ph, jurats isolates,
2. Lipases. Staphylococci produce a number o f
lipid hydrolases which help them in infecting
the skin and subcutaneous tissues.
3. Hyuluronidase breaks down the connective
tissue. Sraphylnkniase (fibrinolysin), forty acid
modifying enzymes and proteases help in
initiation and spread of infection.
4. N uclease. A heat stable nuclease is a
characteristic feature of Staph, auratf.
5. Protein receptors. Staphylococci possess
receptors for many mammalian proteins such
as fibronectindflbrinogenj IgG and C iq . These
facilitate staphylococcal adhesion tu host cell
and tissues,
if 5 s l |j %
C ytolytic toxins arc
membrane-active substances, consisting of four
hemolysins and a leucocidin.
Alpha hejiroJysr/i (Alpha toxin, lysin) is the
most im portant among them . It is a protein
inactivated at 70 °C, but reactivated paradoxically
at 100 This is because at 6 0 -7 0 ;|C 3 the toxin
C o p y rig h te d m aterial
19b * "e x tb c o k
CnrribLn.es w ith a heat labile in h ib ito r w h ith is
denatured at 100 °C, leaving the toc^n fret.
Alpha tn\in Ivscs rabbit erythrocytes, hut is less
active against sheep and human red cells. It is also
leucocldal* cy to toxic, dcrm onccroric [on
irUraderma] inoculation in rabbits), neiiroioxic and
lethal (or: intravenous inoculation in rabbit'1. I l is
toxic to macrophages, lysosomes, muscle tissues,,
renal cortex and the circulatory system.
Befa hcfnntvxin is , l nphingnmyetirtiSe, hemolytic
for sheep cells, but not For human or rabbit cells. It
exhibits a ‘hot-cold phenomenon', the hemolysis
being initiated at 37 °C, but becoming evident only
after chilling.
t j-.j r^jrrj.j hemofysin l> composed of two separate-
proteins, both of which ate necessary for hemalync
activity.
D f/tJ hemolysin has a detergent-like effect on
cell m embranes o f erythrocytes* leucocytes,
macrophages and platelets.
Leucockfjrn (called the Panton-ValcnTine toxin
after its discoverers) is also a two component toxin,
like the gamma Lysln, being composed of two
com ponents (5 and F ). Such hicom ponent
membranc-acfive toxins as the staphylococcal
leucocidin and gamma lysin have been grouped as
s\T}Cfgohymcnotmp\i' toxins.
lin te m to v in : This toxin is responsible for the
manifestations of staphylococcal food poisoning —
nausea, vomiting and diarrhea 2 -6 hours after
consum ing contam inated fond cont.i i r.i ng
preformed toxin. The toxin is relatively heat stable,
resisting 1G0 for 10 to 40 minutes depending
on the concentration of the toxin and n amre of the
medium. About two-thirds of Staph, aureus strains*
growing in carbohydrate and protein food - secrete
the toxin. Meat and fish or milk and milk products
cooked and left at room tem perature after
contamination Wl: 1] staphylococci, tor enough time
for the toxin tu accumulate, are the common items
responsible. The source of infection is usually a
food handler who is a earner. The illness is usually
self limited* with recovery in a day or so.
or M io ro tH o c g y ►
Eight antigenic types ufemerutoxin are currently
known, named A, R, C": jl D, E and H. They arc
formed bv toxigenic strains* singly or m combination.
The toxin is believed to act three tly on the
autonomic nervous system to cause the illness,
rather titan on the gastrointestinal mucosa. The
toxin is antigenic and neutralised by the specific
antitoxin. Type A toxin is respond ilde for most cases.
Sensitive serological tests such as latex
agglutinai ion and ELISA are available for detection
of the toxin.
The toxin is potent, micnogram amounts being
capable of causing the illness. Some cases of post
antibiotic diarrhea are caused hv if
rnterotoxin-
fo rm ing stapbyloCoCci. The toxin also exhibits
p y ro g e n ic, mitogenic* hypotensive, throm bo­
cytopenic and cytotoxic effects.
T o x ic shook syn d ro m e to xin ( T S S T ) :
Toxic shock syndrome (TSS) is a potentially fatal
multisystem disease presenting with fever*
hypotension* myalgia, vomiting* diarrhea, mucosal
hyperem ia and an erythem atous rash which
desquamates subsequently. Tins is associated with
infection o f mucosal or sequestered sites by TSST-
produting S taph aureus strains usually belonging
to bacteriophage group 1. T SS T type-1 (formerly
also known as cntcrotoxin type F or pyrogenic
eaotoxin C) is most often responsible, though
enterutoxins B or C mav also cause the syndrome.
TSS was first identified in 197$ in children and
adolescents, hut became widely known only in 1990
following outbreaks in the USA in menstruanng
women using highly absorbent vaginal tampons,
Theit vag: nal swabs showed heavy growth o f Staph,
aureus, though blood cultures were mvariahly
negative.TSST-1 antihndy is seen in convalescents,
TTii^ is protective and absence of T S S T -1 antibody
is a factor in the pathogenesis of the condition.
Though tampon-related TSS is now rare, the
syndrome occurs in other infections o f rhe skin,
mucosa and other sires and also in some nuigical
wounds.
Staphylococcal enrerotnxins and TSST-1 arc
C o p y rig h te d m aterial
supcn m igcn s which arc potent activators of I
Lymp hocytes. Being V[f restricted T cell nutogens,
such supcramigcns stimulate very large numbers
o f T celts, without relation to their epitope
specificity. T h is leads to an dcdeSiLve and
dysregulated immune response with release o f
cytokines interleukins I, 2, tumour necrosis factor
and interferon gam m a. T h is explains the
multisystem involvement and florid manifestations
in staphylococcal food poisoning and TSS,
T h is
toxin, also known as E f or "exfoliatin' is responsible
for the 'staphylocoCual scalded skin syndrome'
(S S S S ), exfoliative skin diseases in which the outer
layer of epidermis gets separated from the underlying
tissues. The severe form o f 5 5 5 5 is known as
Ri nee's disease in the newborn and toxic epidermal
necrolysis in older patients. Milder tin ms are
pemphigus neonatorum and bullcjut impetigo.
Staphylococcal infections are among the most
common of bacterial infections and range from the
trivial to the fatal. Staphylococcal in Tedious are
characteristic ally localised pyogenic lesions, in
contrast to the spreading nature of strepEOCOCCal
infections. Common staphylococcal infections are
the following:
liilliculi l Ls , furuncle
(boij), abscess (particularly breast abscess), wound
infection, carbuncle, impetigo, paronychia, [css often
cellulitis.
O steom yelitis, arthritis,
bursitis, pyomyosirk.
Tonsillitis, pharyngitis, BinusiuE,
otitis, bronchopneumonia, lung abscess, empyema,
rarely [ineumonia.
Abscess,
meningitis, intracranial thrombophlehitis.
Bacteremia, septicemia, pyemia,
endocarditis
Staphylococci are uncommon in routine
urinary tract infections, though they do cause
infection in association with local instrumentation,
im plants or diabetes. Urinary isolates of
staphylococci ate to be considered significant even
with low colony counts, as they may be related ro
bacteremia.
Staphylococci mav be typed, bated OH (heir
susceptibility to bacteriophages. An internationally
accepted o f phages is used for typing-
Staphylococcal phage tiping is done by a pattern
method, The strain to he typed is inoculated on a
plate of nutrient agar to form a lawn culture. After
diving, the phages are applied over marked squares
in a fbted dose (routine test dose). After overnight
incubation, the culture will be observed to be lysed
by some phases bur not by others. The phage type
of the Strain is expressed by the designations-of all
the phages that Lvsc it. Thus, if a it ruin is Ivscd
only by phages 52. 79 and SO, it is. called phage
type 52779/SO, Phage typing is of great importance
in epidem iological studies o f stuphvfococcal
infections*
international basic set of phages lor typing
Staph, aureus of hymen origin
Group I 29, 52, S2A, 79,80
Group U 3A . 3 C . 5F, 71
Group ]] I 6 , 42E, 47, S3, 54, 75, 77, 83A,
84, 85
Group IV
Group V H 96
Not all oca ted 81, 95
Nor all cultures are typable by this procedure,
and rhe susceptibility patterns of circulating strains
vary in rime and locality. Hence phages in die
reference scr require periodic revision.
StaphyLocorci are primary
parasites o f human beings and animals, colonising
the skin.^kin glands and mucous membranes. The
most common sources of infection are human
paticIIrsund carrier^ animals and inanimate objects
being less important. Patients with superficial
infections Hind respiratory infections disseminate
C o p y rig h te d m aterial
19G * Texlbook ol Microbiology ►
large numbers o f staphylococci into the
environment- About 1 0 -3 0 per cent of healthy
persons cany staphylococci in the nose and about
10 per cent in the perineum and also on the hair.
Vagina! carriage o about 5 -1 0 per cent, which rises
greatly during metises a factor relevant in the
pathogenesis ofT S S related to menstruation.
Staphylococci] carriage starts early in Life,
colonisation o f the umbilical stump being very
common in babies bom in Hospitals. Some carriers,
called Lshedders', disseminate very large numbers
of cocci for prolonged periods- The cocci shed by
patients and carriers contaminate fomites such as
handkerchiefs, bed linen and blankets and may
persist on them for days or weeks. Staphylococci
may also come from infected domestic animals such
as cows.
Staphylococcal disease may follow endogenous
or otogenous infection- The modes o f transm it ion
may be by contact, direct or through fomites, by
dust or by airborne droplets.
Hospital infections by staphylococci deserve
spec id attention because of their frequency and
because they arc caused by strains resistant to
various antibiotics. Staphylococci are a common
cause of postoperative wound infection and other
hospital cross infections. Most of these are due to
certain strains of staphylococci that are present in
the hospital environment, the so-called 'hospital
strains1. They belong to a limited number of ph;gc
types and are commonly resi-itant to penicillin and
other antibiotics routinely used in hospitals. Some
of them, the ‘epidemic strains', cause epidemics of
hospital cross infections. The first of these to be
recognised was phage type Sfl/Sl, which accounted
for most of Staphylococcal infections in hospitals
throughout the world- They have since been
replaced by other stra ins of staphylococci and by
Gram negative bacilli.
Measures for the control o f staphylococcal
infection in hospitals include:
1. isolation of patients with open staphylococcal
1l-sii
Z detection o f staphylococcal lesions among
surgeons, nurses and other hospital staff and
keeping them away from work till the lesions
are healed;
3, strict aseptic techniques in theatres;
4, the oldest, simplest and the most effective method
of checking hospital cross infection is hand
washing, which unfortunately is often
neglected.
If an outbreak o f staphylococcal sepsis ucCurs,
a search may be made for carriers among the
hospital ‘■t ;lA . THqsl1detected should be treated with
local applications of neomycin and chlorhexidine.
In some institutions in America, eradication of the
virulent resident strain has been attempted by the
deliberate disseminari on of a strain o f low virulence.
The latter may oust the former by interference.
Antimicrobial prophylaxis by topical applications
o f antiseptics such as hcxachtcuophcnc has also
been found useful.
L a b o r a to r y diagnosis: The specimens to be
collected depend on the type of lesion (for example*
pus from suppurative lemons, sputum from
respiratory infections). In cases of food poisoning,
foces and the remains of suspected food should be
collected- For the detection of carriers, the usual
specimen is the nasal swab. Swabs from the
perineum, pieces of hair and umbilical stump may
be necessary in Special situations.
Direct microscopy with Gram st.dried smears
is useful in the case of pus. where cocci in clusters
may be seen. This is o f no value for specimens like
sputum where mixed bacterial flora arc normally
present.
Diagnosis may readily be made by culture. The
specimens are plated on blood agar. Staphylococcal
colonics appear after overnight incubation.
Specimens where staphylococci one expected to be
scanty and outnumbered by other bacteria (for
example, swabs from earners, feces in food poisoning
cases) are inoculated on selective media like
Ludlanfs or salt-milk agar or Robertson's cooked
meat medium containing 10 per cent sodium
C o p y rig h te d m aterial
 - - 1
• - -

chloride. Smears me examined £rum the niituAs

and the eoagulssr rest done when staphylococci
ire isolated.
Coagulate test lh dune by two methtMln — mhe
and hJilIc cnaguJase Eesls. The tube mggulasr test
detects free cwigulivc, ^ k n t 0-1 ml o f a young
broth culture or agar culture suspension o f the
isolate is added lo about <1.5 nil o f htunan Lit rabbit
plasma in a narrow test tube. ED TA , oxalate or
heparin may be used as the anticoagulant for
preparing the plasma. Citrate is not recommended
because at may he utilised by some contaminant
bacteria, causing I . i I - l positive result h . Positive and
negative Controls are also set up. The tubes are
meubated in a water bath at 37 °C for 3—6 hours. If
positive, the plasma clots and does nor flow when
the tube is tilted. Continued incubation is not
recommended as the dot may get Lysed by the
fibrinogen formed bv some strains.
The slide rest detecting bound coagulate is much
simpler and usually gives results parallel with the
rube test- When there is divergence, the tube test
will Ik t!te deciding factor. Foi the slide test, the
isolate is emulsified in a drop of saline or a slide.
After checking tor absence of autoagglud nation, a
drop of human or rabbit plain a is added to the As drug resistance is to com m on among
emulsion and mixed. Prompt clumping of [he cocci staphylococci, the appropriate antibiotic should be
indicates a positive test. Positive and negative chosen bated on antibiotic sensitivity tests. Benzyl
controls also art scr up. penicillin is the most effective antibiotic, if the
Antibiotic sensisiviry tests should be performed strain is sensitive. M ethicillin was the first
as a guide to treatment. This is important as compound developed to combat resistance due to
staphylococci readily develop resistance Us drugs, penicillinase (beta Lactamase) production by
Bacteriophage typing may be done if the staphylococci. Due to the limitation!; in clinical use
information i;. desired i<it epidemiological purposes. of mt;r liifil Iin, cloxacillins are used instead against
Other typing methods include antibiogram pattern, pentdUmast-producing Strains. But methicillin
plasmid profile, DNA fingerprinting, ribotyping resistant srrains o f £fiiph. aurcitf (MRSAj became
and PCH-biied analysis for genetic pleomoiphism. common, which were resistant not merely to
Serological tests may sometimes be of help in penicillin, bur also to all other beta Lactam antibiotics
the diagnosis o f h id d e ri deep in fe c tio n s , and many others besides. For life threatening
Anriitapbyiolyiin (anti alpha lysirt) titres of more staphylococcal infections, vancomycin is the drug
than two units per ml, especially when the title is Lit choice. Strains resistant tn vancomycin and
rising, may be of value in The diagnosis of deep Icicoplanin have appeared in hospitals where
seated infections such as bone abscesses, antibiotic use is indiscriminate, For mild superficial

C o p y rig h te d m aterial
Iflion^ antibiotics may not be necessity
Ibpiral applications ofdrugs not used svsLenncally,
;ls bacitracin, chlorhcxidiiw or mupirocin may he
suffiOCn t.
Some strains show the phenomenon of drug
tolerance. These strains will lie found to be
susceptible in the disc sensitivity test but their
minimum baetc ric]dal concentration will be very
rnuuh higher than their minimum inhibitory
concentration. They lire emt killed hv antibiotics nl
the usual doses and persist, leading to failure in
eradicating the infection.
The treatment of carriers is bv local, application
d1 suitable Antibiotics such as bacitracin and
antiseptics such as chlorhcxidine. In resistant cases
posing major problems, rifampicin along with
another oral antibiotic may he effective in long term
suppression or elimination of the carrier state.
Ik Hides Sftiph. aureus, a few other staphylococcal
species are coagulase positive, e.g. Staph,
intcrnicxiiux and Staph, hyiewi. These are animal
parasites and do Out ink'd huruins.
Coagulate negative staphylococci const i.Eutc j major
component of the normal flora oi the human botlv.
Some species of coagulasc negative staphylococci
eau produce human infections - Staph, fpitfcrmidlis,
■Sfjpb, haemofyticus and Staph, taprophyticus.
Staph, cp id om id is is invariably present on
normal human skin. It is nonpat hogenic ordinarily
C o iju h ic 4 -
Novobiocin Sensitivity S S
Acid from mannitol anaerobic ally 4 ■
Phosphatase
but can cause disease when the host defences are
breached. It is a common cause of stitch abscesses.
It has i predilection for growth on iinplanfed
foreign bodies such as artificial heart valves, shunts,
intravascular catheters and prosthetic appliances,
leading co bacteremia. Hospital strains of Stmph.
cpidctm idis sue usually multiple drug resistant. It
can cause cystitis. Endocarditis may be caused,
particularly in drug addicts.
Staph, sappophytiats may he present on normal
human skin and the periurethral area and can cause
urinary tract infection, particularly iti sexually active
young women. The mfecdon is symptomatic and may
involve the upper urinary tract also. Men are
infected much (css often, though it is sometimes seen
in older persona. The infecting strains arc usually
sensitive to most com m on antibiotics, except
nalidixic acid. Stiffih. aaprophytioa is novobiocin
resistant.
Table 2 2 .1 lists the features useful for
distinguishing the major Specie* uf ^taphylncocd.
These ife Gram punitive cocci which occur nmstlv
in pairs, tetrads or irregular clusters. They arc
catalase and OJcidase positive. They are aerobic with
a strictly ic-spiratorv metabolism. They are parasitic
on mammalian skin and are ordinarily
nonpnithogcnic. They resemble staphylococci but
in stained smears the cells are generally larger and
more Gram variable than staphylococci. In cultures
they form smaller colonics. The common laboratory
test used to differentiate between micrococci and
-
R
4 -
C o p y rig h te d m aterial
 4 Stsp^ylococcuE f 201

staphylthiocc1. is the Hugh and. [jtLifRnn':i oxidarion— oxidative and staphylococ.'. show ftrmcntariyc
ferm entation rest in which m icrococci show patterns.

F u rth e r It ending
Cmrtley KB and GC Archer. 1997. SlaphyltKwat human disease. Nrw York. Ctiurchill-LiviiigbLcim;.
Easmon. CSF and C Adlani 1983. ttm! SiaphviwtKiwi Infections : London: Academic Press.
K . : h i l . W E , J r i c l T L " L i i v . : : i i i . l i i 1 9 9 4 . C . 11. i .i . - . ^ n d iL 'a n c E : ■:I G c q g u lu x c r u g . i l i v r i. L J in . A J . 'r . '- jlu . u' j J i ''.1 7 : 1 1 7 .

Manf#LLk P and J ECapple 1990- StaphylnciiccR] entercHo'ins and tlr ir relative?, Stance 248: 705.
Rupp ME and GL Aichcr 1994. Coagulate nqgjftn acajshyLotuiid, AmJ Sitd 9 4 :313.
ShtapCrtJN. 1954. 5l,.| !^ Iiko. , h1- iLLfcm ~ tin- persMcnl p.i'hiiLi. n. iVcw P-nfiiJ jVletf, 310, 13^8t ]437.

C o p y rig h te d m aterial
 Streptococcus
Streptococci are Gram potitirc coed arranged in
chains or pairs (Fig. 23.1). They are- part of the
normal flora of humans and an imath. Some of them,
arc human pathogens, 'The most important of them
is S tr e p to c o c c i* pyogcnen causing pyogenic
Infections, with a characteristic: tendency to spread:,
as opposed to staphylococcal lesions which are
typically localised. it is also responsible for the
nonsuppLirarive lesions, acute rheumatic fever and
glomerulonephritis which occur us sequelae to
infection.
D m in chains were first Seen in erysipelas and
w ound infections hy Billroth {18 7 4 ), who called
them streprococci (srrepfos, m eaning twisted or
Coiled). QgStoJt (lSSl) isolated them from acute
.Lbscesses, distinguished them from staphvtococci
and established their pathogenicity by animal
inoculation. Roscnbach (1684) isolated the cocci
from human suppurative lesions ami gave them the
name Stftptacoocw pyogenes,
, ■: •
’ -.V, - ’
-%j* 3, -I ■ k j* / J
Several systems of classification have been employed
but in medical bacteriology the billowing method
is useful {Fig. 2 3 .2 ).
Streptococci are first divided into obligate
anaerobes and facultative anaerobes. The former
are designated pepiostreproeocci and are considered
in a later chapter. The aerobic and facultative
anaerobic streptococci arc classified on the basis of
their hem olytic properties. Brown (1 9 1 9 )
categorised them into three varieties based on die ir
growth in 5 % horse blood agar pour plate culture-..
Alpha (a) hemolytic streptococci produce a
greenish discolouration with partial hemolysis
around the colonics. The zone tjf lysis is small (1
or 2 mm wide) with indefinite margins, and unlysed
erythrocytes can he made out microscopically
within tins zone. These are known as Viridans
streptococci' or Streptococcus viridans (from
lviridisr meaning green). The alpha streptococci are
normal commensal s in the threat, but may
cause opportunist infections rarely. Pnemococcue
pn eu m on iae) is also an alpha hemolytic
streptococcus.
B e t a ([3) hemolytic streptococci produce ft
sharply defined, dear, colourless zone of hemolysis,
mm wide, within which red cells are
C o p y rig h te d m aterial
 n.XTHOOK fH- MICKOblOUXil
Stri-plikiiffl

(J; tc^uhcttkdC

Afrobe> UK) rstullsI i f Ohllgjn: i^cKiK'--

nnscrutiT) Pe pfemsrs ptsicflct i
„ 1
Httwlyais
J

*
j Alpha .1 i linim.i
Mbs pt«jp:i (The cnerrkovj;lis giogp)
C lugified uiir. qfiKJf» hs phyiiu- n±-.-:l'ii.,il inc.- spetn!’. h, phy-.iiv
Ihgical >nl bjkVticIBH.'sJ pr«ipcrtli;t b-ipt il and htrtclK*n*£*J prcptriici

2. Bi-U
(The hcmcilyltc ilneptoLoccij

Stffuili^iCa CfOup’■petrfw.' t‘ Ldrh- 'htdralf aruig«*i

2D L lK t f r ld Grwp» lABCDEJKrhlKL
MVOKJKSTLV.
Group A - JirifJMneoi'dhS pwjt/iWJ

Sc id lexical i\ pi (M fliM rinJ

fJnHkh <>pw(I, 2.3y(«,>

completely lysed. The term ^hemolytic streptococci'
strictly applies only to beta lytic strains. M ost
pathogenic streptococci belong to Lht> group.
Gamma (?) or nonhemolytic streptococci
produce no change in the medium and so arc
sometimes referred to as ‘in difiavn t stnyVtxoca-
Tbey include the fecal streptococci (eonemcoea,
Str, /accaj/ff) and related species, They are called
the 'enterococcus group'.
Hem olytic streptococci were classified by
Lancefield (193d) serologically into groups based
on the nature of a carbohydrate [C) antigen on the
cell wall. These arc known as Luncefield groups,
twenty o f which have been identified so far and
named A -V (without 1 and J). The great majority
of hemolytic streptococci that produce human
infections belong to group A . H em olytic
streptococci of group A arc known as £lr, pyogenes.
These may be further subdivided into types based
on the protein (M , T and R) antigens present on
the cell su rfacc (Griffith typing). About eighty types
of Srr. pyogenes have been recognised so far (types
1, 2, 3 and so on).
Table 2 J.1 shows the medically important
streptococci and their characteristics.
STREPTOCOCCUS F K i S ^
The individual cocci are spherical
or oval 0 .5 -1 .0 pm in diameter. Size variations
result from cultural conditions, for example, when
grown anaerobically they are somewhat smaller.
They arc arranged in chains, the length of w hich

C o p y rig h te d m aterial
 P .>r.Li.-lV1i*l T.jT _
■■;,ii' K

iSfr, pyqgrnes A beta Tuout, llafitra-dn sensitive; Upper respiratory tract

skin PYR tesi positive; infection*; pyoderma;
Riitose not fermented rheumatic fevo;
glmnetulonephriris

Str. §g?I#fbac B beta Female CAM!3 test, hippuratr Neonarsl mcningins,

genital hydrolysis *epticcmU
tract,
rectum
Str- eqiitiimiiis C beta Throat Rihose and trehaloEe laryngitis,
fcTnncntarion endocarditis
Sir. ajt£irto?u$ A,C,K,G, ben (alpha, Throat, Group A strains Pyogenic infections
untypable gamma) colon, hacitiacin resistant
female PYR negative; Minute
genital colony variants of
tract inrfiL-f groups
tiitETWOlj ip. L> Gamma Colon Growth in 6-5% NaCI; Urinary tract
(Str. faccalis and (alpha, PYR positive infections,
other enterococci) beta) endocarditis,
suppurative infections
NonentcrotOCtal l> gamma Colon No growth in 6.5% Endocarditis
Group D species NaCI
(Str. bovis}
Viridans Not typed alpha Mouth, Oprochin resistant, Endocardi tis ( Str.
streptococci {many (gamma) ooIon, species classification sanguis); dental caries
specie*) female on biochemical i 5tr. FilcrOU^
genital properties
tract

varies within wide limits and is influenced by the
nature of the culture medium, chains being longer
in liquid tlum in solid media. Chain formatniil i&
due to the cocci dividing in nnc plane only and the
daughter cells filing to separate completely. ITkcrr.-
is often an appearance ot pairing within the chains.
Significance was o n « attached to the length nf
the chains, md streptococci had been classified
accordingly (S et. irjj^tts and b m is) but this has no
relevance to vimlence nr other properties. III fact,
some norpathngcnic streptococci form the longest
chains, for example. Sir. sitivirius. (Fig. 23.1).
Streptococci arc nonmotilc and nonsporing.
Some strains of Str. pyogenej and some group C
strains have capsules composed o f hyaluronic acid,
while polysaccharide capsules are encountered in
member:- of group U and D. These capsules are
best seen in very young cultures,
Jt is an aerobe and
a (acultarive anaerobe, growing best at a temperature
o f3 7 *C (range 22-42 ^C). It is exacting in nutritive
requirements, growth occurring only in media
containing fermentable carbohydrates or enriched
wiiti blood or serum. On blood agar, after incubation

C o p y rig h te d m aterial
 * Streptococcus
for 24 hours, the colonics are small {0.5—1.0 mm)
c ire u la r, seirn'ransparent, tow c o n i'e x dihii.3 with un
area o f dear hemolysis around them. Growth and
hemolysis are promoted by L0 per cent C O a.
Virulent sit.llm>, ou fresh isolation from lesions,
|■i ■ I l- a m att (finely granular) Colony, while
avirulent str.iins form "glossy" colonies. Strains with
well marked capsules produce ■mucoid’ colon cs,
corresponding in virulence to the matt type. Very
rarely. nonhemolytic group A streptococci are
encountered, which are typical of Str. pyagenex in
other respects.
Tn liquid med::insuch as glucose or scroro broth,
growth occurs as a granular turbidity with a
powdery deposit. No pellicle is formed.
Etioohem ioal re a ctio n s: Streptococci ferment
several sugars produ. i:iL acid hut no gas.
Streptococci ire catalase re stiv e . They are not
fioluhle in 10 per cent bile, unlike pneumococci.
Hydrolysis of pyrrolulonyl naphthylamide (PYR
test) and failure to ferment rihose are useful in
differentiatuig S'u; pvu^nes from ocher streptococci.
R e s is ta n c e Str. pvr^encs rs a delicate organ i-m,
easily destroyed by heat [54 "G for 30 minutes). It
dies in a few davs v in cultures,* unless stored at a low
temperature (4 "C l, preferably in Robertson's
cooked meat medium. It can, however, survive in
dust for several weeks, it protected from sunlight-
It is rapidly inactivated by anrscptics. It is more
resistant to crystal violet than many bacteria,
including Staph. aureus. Crystal violet (1 tng/L),
nalidixic avid {15 mg/L) and colisrin sulphate (10
mg/T.) added tn blood agar provide ,l l;-!mmLselectLve
medium iortht isolation of streptococci, including
pneucnucocc-i. It is susceptible to sulphonamides
and many antibiotics but unlike Staph, aureus does
n.it develop n-sL^r.i n. to drugs. Sensitivity to bacitracin
is employed as a convenient method fordiffcrenri.uing
Str. pyogenes from ocher hemolytic streptococci.
A n tig e n ic stru c tu re : Fig. 2 3 ,3 illustrates the
disposition of the various antigens in Str. pytygenes.
The capsule when present inhibits phagocytosis.
It is not antigenic in human beings.
* 205
The cell wall is composed of an outer layer o f
protein and [iputeichoie .i-.n.l, a n-.iddle layer of
group specific carbohydrate and an inner layer o f
pepddoglycin.
The pepddoglycan (mucopmtein) is responsible
for cell wall rigidity. It has also some hiulogical
properties Ruch sr pyrogenic and thrombolytic
activity.
Serological grouping of streptococci depends on
the C carbohydrate, Str. pyugenes belongs to group
A. A r th is antigen is an integral part o f the cell
wall, it has to be extracted for grouping by a
precipitation test with group antisera- For the test,
streptococi i are grown in Todd-Hewm broth and
extracted v. ith hydrochloric acid (Lancefield's acid
CX tract ion m ethod), or form amide {Fuller's
method) or by an enzyme produced by Streptontyee^
albus {Maxted'r method) or by autoclaving ( Rantr
and Randall's method). The extract and the specific
anil sera are allowed to react in capillary tubes.
Precipitation occurs within five minutes at the
interface between the extract and the homologous
antiserum. Grouping may also be done by agar gel
precipitation.
Several protein antigens have been identified in
the outer part of the cell wall, Str jm gcjics can be
typed, baRed on the su rfa ce proteins M, T a n d R.
The M protein :s the most important of these. It
acts as a virulence factor by inhibiting phagocytosis.
It is anti.i^enic.The antibody to M protein promotes
phagocytes ? o f the coccus and is therefore
protective. The M protein is hear and acid stable
b u t suscep tib le t o trypt..' d i g e s t i o n . Jt can he
extracted by the Lancefield acid extraction method
and typing is done with type specific sera. About
SO M protein types have been recognised.
T h e f prate ii- is an avid labile, trypsin resistant
antigen present in many serotypes of 5m pyqgcncs'-
k may be specific but many d-fferent M types
possess the same T antigen, lc is usually
demonstrated by the slide agglutinatii- rn test u-ing
trypsin-treated whole streptococci. Some types of
Str, pyogenes (types 3. 3, 28 and 48) and some
C o p y rig h te d m aterial
strains of groups B, C and G contain a third antigen,
the R protein. The T and R proteins have no relation
to virulence. A rutn^pe-tpedtic protein, associated
with the M protein, has been identified. This is
known as M associated protein (MAR).
Hair-like pili (fimbria) project through the
capsule o f group A streptococci. T h e pLLi consist
partly o f M protein and arc covered with
liputeichoLC acid w hich is im portant in th e
attachment of streptococci to epithelial cells,
Various structural components of StT- pyogenes
exhibit antigenic cross reaction with different
tissues of che human body. Antigenic relationships
have been dem onstrated between capsular
hyaluronic acid and human synovial fluid, cell vvj.ll
protein and myocardium, group A carbohydrate and
cardiac valves, cytoplasmic membrane antigens and
vascular intima, and peptidoglycans and skin
antigens. It has been postulated that these antigenic
cross reactions may account for some o f the
manifestations of rheumatic fever and other
streptococcal diseases, the tissue damage being of
an immunological nature
2*
• La
ae
Iffr cii .Jv “4‘-,
Hi ft Ok-iff S t DI Il31p fe :i7y|
jpHJTp' K^i'SMuS •>'/' Irffipfc'tfi
ft.-ft: i-J's' iteilMr'- ft
fjQm&i* ft. \M !>i:::>•> -;ftVi
Str.
pyogenes forms several exotOVtns and enzymes
w hich Contribute to its virulence. Besides these,
the M protein also act? us a virulence factor by
inhibiting phagocytosis, 'fh c C polysaccharide has
been shown to have a toxic effect on connective
tissue in experimental animals.
Strep tococci produce two
hemolysins, streptolysin ‘O' and 'S'. Streptolysin
O is so called because it is oxygen labile. It is
inactive in the oxidised form but may be
reactivated by treatment with mild reducing agents.
Ort bkmd agar, Streptolysin O activity is seen only
In pour plates and not in surface cultures. It may
be o b ta in e d in the a ctiv e state by growing
streptococci in broth containing reducing agents
such as sodium hydrosulphite. It is also heat labile.
It appears to be important in contributing to
virulence. It is lethal on intravenous injection into
animals and has a specific cardiofoxic activity. It
has kucotoxic activity also. In in biological action,
streptolysin O resembles the oxygen labile
hemolysins of CL perfrmgem, Cl. retard and the
pnCu mcHJOccus.
Streptolysin O is antigenic and antistreptolysin
O appears in sera following streptococcal injection.
Estimation of this antibody (ASO title) is a standard
serological procedure for the retrospective diagnosis
of infection with Srr. pyogenes. The Lytin is
inhibited by cholesterol hur not by normal sera,
Followiiig certain chemical treatments or bacterial
contamination, sera may develop inhibitory activity
due to some changes in the lipoproteins. Such sera
arc unfit for the A S O tesr, Because of the
complexity of the hemolysis inhibition test, ABO
rest is now done by the serological method o f latex
agglutination. An ABO litre in excess of 200 units
is considered significant and suggests either recent
or recurrent infection with streptococci.
Streptolysin S and O are produced by groups A, C
and G also.
Streptolysin £ is an oxygen stable hemolysin and
so is responsible for the hemolysis seen around
C o p y rig h te d m aterial
streptococcal .colonics on the surface of blood agar
plates. Il ls called streptolysin S since it is soluble
in serum. It is a protein but is not antigenic.
Convalescent sera do not neutralise streptolysin S
activity. It is inhibited nonspecifically by serum
lipoproteins.
T h is toxin was
named 'erythrogeiue1 because its in ir adeems!
injection into susceptible individuals produced an
erythematous reaction (Dick testH1924), This test
was used to identify children susceptible to scarlet
fever, a type of acute pharyngitis With extensive
erythematous rash, caused by the Sir. pyogenes
strains producing this toxin, Hunching- of the rash
on local injection o f convalescent serum was used
as a diagnostic, test lor scarlet fever (Schultz.
Charlton reaction, 1 18). The Dick test and Schultz
Charlton reaction are now only of historical value
as scarier fever is no longer a common or serious
disease.
The primary effect of the toxin is induction of
fever and so it was renamed Streptococcal pyrogenic
exotoxin (SHE). Three types o f SPE have been
idenrilteid=SPE A rR and C. Tvpes A and C are
coded for by bacteriophage genes, while type B
gene is chromosomal. SPEs ate ‘supefantigens’ (like
staphylococcal cnteroDoxins and TSS toxin), T cell
m itogens which induce massive release o f
inflammatory cytokines causing fever, shock and
tissue damage-
T h is toxin
promotes the lysis of bumail fibrin clots- bv activating
a plasma, proeuxsor (plasminogen). It an antigenic
protein and neutralising antibodies appear in
convalescent sera, Anti-streptokinase anribody
provides tetro-s|iective evidence o f streplociHfCal
infection. 1 ibrinoly^in appears to play a biological
role in streptococcal infections by breaking down
the fibrin harrier around the lesions and facilitating
the spread of infection- Streptokinase is given
inrravc nousiy for the treatment of early myocardial
infarction and other thromboctnboloic disorders,
These cause depolymerisation o f
DNA. Pyogenic exudates contain large amounts ul
DNA, derived from the nudd of necrotic cells.
Strcptodornase helps to liquefy rhe thick pus and
tnav he responsible for the thin -serous character of
streptococcal exudates. This property has been
applied therapeutically in liquefying localised
collections o f thick exudates,, as in empyema. A
preparation containing streptokinase and
stiepiodornase is available for this gvurpose. Four
an tigjenically distinct I JNAises-, A„ R, C and D. have
teen recognised, of which type B is the most
antigenic in human beings. Demonstration o f ami-
DNAase h antibody is useful in the retrospective
diagnosis of Sn. pyogenes infection, particularly in
skin infections, where ASO fitres may be low.
Streptodomase ft and 1) also possess rihonudease
activity,
i
This acta on the
cocnzvme NAD and liberates nicotinamide from
the molecule, it is antigenic and is specifically
neutralised by the antibody in convalescent sera.
The biological significance o f CVADasc is not
knowrtj though it is believed to be leUCOtOxic,
This enzyme breaks down the
hyaluronic acid of the tissues. This might favour
the spread of infection along the intercellular spaces.
Streptococci possess; a hyaluronic acid capsule and
also elaborate a hyaluronidasc— a seemingly sclf-
destructivu process, It is, however, found that strains
that form hyaluroEiidasc in targe quantities (M types
4 and 2 2 ) are noncapsulated. The enzyme is
antigenic and specific antibodies appear in
convalescent seta.
Some hi types of Str.
pyogenes produce a lipoproccinasc which produces
opacity when applied to a ^ r gel containing horse
or swine serum. This is known as scrum opacity
factor (SO P).
M a ry strains alsn produce proteinase.
C o p y rig h te d m aterial
m ^ TeKitHo-. of Microbiology *
phosphatase* esterases, amylase, N acetyl
glucusaminldase, neuraminidase and other toxins
OT euzvnncs- 1■ Is not known whether, and to what
extent, these contribute to pathogenesis.
P VTH OtiEN IClTl
Str pvogcncs produces pyogenic infections with a
tendency to spread locally, along lymphatics and
through the hloodstreum.
R esp irn iory in fectio n s: The primary sire of
invasion of rhe human body by Sfn pyqgenes is the
throat. Sore th ro at Is the m ost com m on of
streptococcal diseases. It may be localised as
ton-hilliiis nr may involve the pharynx more diffusely
(pharyngitis). Virulent group A streptococci adhere
to the pharyngeal epithelium by means o:
lipoteichoic acid covering tbe surface pili. The
glycoprotein fibroncctin on the epithelial cells
probably serves as the lipoteichoic acid ligand.
Tonsillitis is more common in older children and
adults than in younger children, who commonly
develop di ffuse pharyngi ri-i. Li icaiisar ion is be! ieved
to be favoured by hypersensitivity due to prior
contact.
From the throat, streptococci may Spread 10 the
surrounding tissues, leading to suppurative
complications, such as otitis media, mastoiditis,
quinsy, Ludwig's angina and suppurative adenitis
I t may rarely lead to meningitis, Streptococcal
pneumonia seldom follows lliroal infection but m.iv
occur as a complication of influenza nr other
respiratory viral diseases.
S k in a n d s o rt tis s u e in f e c tio n s : Str,
pyogenes causes a variety of suppuratuT infections
of the skin. Including infection of wounds or bums,
with a predilection to produce lymphangitis and
cellulitis. Infection of minor abrasions may at Limes
lead to fatal septicemia.
The two typical streptococcal infections of the
skin are erysipelas and impetigo. The former is 1
diffuse infection involving the superfiL-i.Ll
lymphatics. The affected skin, which is red, swollen
and indurated, is sharply demarcated from the
surrounding healthy area. While erysipelas is rue
and seen only in older patients, impetigo is found
mainly in young children. Impetigo is caused by
Str. pyogenes belonging to a limited number o f
serotypes, usually the higher numbered M types,
iustead of the lower numbered M types wl Livh cause
throat infection*. Impetigo and streptococcal
infection of scabies lesions arc rhe main causes
leading to acute glomerulonephritis in children in
the tropics.
In pyoderma, antibody response to streptolysin
0 is not high and ASO estimation does not have
as much clinical -.igniffcancc as in pharyngeal
in fe ctioits. Antibody to D N A ase B and
hyaluronidase arc more useful in retrospective
diagnosis o f pyoderma antccendcnt to acute
glomerulonephritis.
Streptococcal subcutaneous infections range
from cellulitis to necrotisiog fasciilifi. The latter
condition is more commonly caused by a mised
aerobic aRd anaerobic bacterid in lev Lion but some
strains of Sm pyogtne s (more particularly M types
1 and 3 strains forming pyrogenic exotoxin A) may
alone be responsible- This is ordinarily a sporadic
condition and has been known since 1883 but small
outbreak in the UK and the USA have recently
caused much alarm because of their seventy and
high fatality. These str.ii.nS have earned notoriety
under the name 'flesh caring bacteria*. In such cases,
extensive necrosis of subcutaneous and muscular
tissues and adjacent fesci.i is associated with aseVrfc
systemic illness— a toxic shuck-likc syndrome with
disseminated intravascular coagulation and multiple
system failure, £ rr pyogenes can be isolated from
the affected site and rising lilies o f antistreptolysin
and anti ‘DNAase B demonstrated- Though the
isolates are penicillin sensitive in vitro, treatment
with pemdHin may not be effective, Vancomycin
is the drag of choice in life threatening infections.
Soft tissue Infections with lom t M types o f S n
pVT^rjTes(1, 3, 1^, 2W) may sometimes cause a tuitiv
shock syndrome resembling staphylococcal TSS,
Streptococcal TSS and necrotizing fasciitis occur
C o p y rig h te d m aterial
 * SUepto coccus ►
only in persons nnnimmune u> the infecting M
types.
G e n ita l in fe o tio n s: B oth aerobic and
anaerobic streptococci are norma] inhabi;ants of
the female genitalia. Str.pyngenesvfns an important
cause of puerperal Rep-.is, with the infection usually
being exogenous. The emphatic demonstration by
Semmelweis in 1847 that hospital outbreaks of
puerperal fever could be prevented by the simple
measure of handwashing by those attending the
labour wards remains a landmark in clinical
microbiology. Puerperal fever is now much more
commonly due to endogenous infection with
anaerobic streptococci. Streptococcai puerperal
sepsi-i u-Sed 11:■ take a heaw tot! o f lile before
antibiotics became available.
< )iiie r su p p u ra tiv e infecti^n A t Str.
pyogenes may cause abscesses in internal organs
such as the brain, lungs, liver and kidneys, and also
septicemia and pyemia.
N o n s u p p u r a tiv e e o m p lio a tn jn s t S fr
p y og en es infections lead to two im portant
nonsuppuralnfe sequelae— acute rheumatic fever and
acute glomerulonephritis. These complications
ensue J -3 weeks after the acute infocrion so that
the orgac.ism may not be detectable when sequelae
set in. They differ in their natural history in a
number of respects ('fable 23.2).
The pathogenesis of these complications is not
clearlii understood. The essential lesion in rheumatic
fever is carditis, including connective tissue
degcneraf.on of the heart valves and inflammatory
m yocardial le s io n s characterised by A schoff
nodules. Typi cally, rheumatic fever follows persistent
or repeated streptococcal throat infections with a
strong antibody response. The lesions are bdheved
to be the result of hypersensitivity to some
streptococcal component. It has also been suggested
that there may be an element o f autoimmunity
involved, and anrigcnic cross-reactions have been
demonstrated between streptococci and heart
tissues. Lesions resembling rheumatic fever have
been produced experimentally in rabbits by repeated
209
infection with pyqgenesand in mice by inaction
of sonic tysafes of the coo. i.
W hile rheumatic fever may follow Infection
with any serotype o f Str. pyt-genes, nephritis is
caused by only a few 'ncphritogrnic' types. In the
tropics, shin infections are perhaps more important
in this respect than throat Lnfectinns. TTte nephritis
is usually a self-limited episode that resolves
without any permanent damage. The pathogenesis
may be due to antigenic cross-reactions between
the glomerular membrane antigen and cell
membranes of ncphritjogcnic streptococci, or more
often it may be an immune complex disease. This
condition has been produced in monkeys and rabbits
by repeated infection with type 12 Str. pvra genes or
injection of bacterid products, and in mice with
soluble streptococcal products.
E p id e m io lo g y : T h e m ajor source o f £fr,
pyogenes is the human upper respiratoty tract -
throat, nasopharynx or nose - of patients and
earners. Carrier rates o f up to 20 per cent have
been observed, Symptomiess infection is common
and helps to m aintain the organism in the
community. Transmission of infection is cither by
d incct contact or through contaminated fingers, dust
or fbmltes. In the tropics, streptococcal infection
of the skin is common and may be spread by
nonbiting insects, particularly the eye gnat
Hippelates.
Streptococcal infections a f the respiratory tract
arc more frequent i:i children 5 - 8 yean o f age than
in children below two years or in adults. They are
more common in winter in the temperate countries.
No seasonal distribution has been identified in the
tropics. Crowding is an important factor in the
transiti iss ion ul infection. Outbreaks of mfeCLion
may occur in dosed communities such as hoarding
schools or army camps.
Immunity is type specific and appears to be
associated with antibody to the M protein.
Reinfections occur because of the multiplicity of
serotypes.
L a b o r a to r y d ia g n o sis; In acute infections,
 Site ot infection Throat
Prior senaitwatiun EFBential
Semiypt of A IIY
Str. piiqjenes
Immune response Marked
Complement lo c i Lrn.ittccted
Generic susccpriluliry Present
Rtftited STracks Common
Penicillin prophylaxis Essential
Course Progressive or static
Prognosis Variable
diagnosis is established by culture, while in the
nonsuppurative complications, diagnosis is mainly
based on the demonstration of antibodies.
Presumptive information may be obtained by an
exami nation ot drum stained films- from pus and
CS F. The presence ot t Jram positive cocci i n ctiai ns
is indicative of streptococcal iniection. However,
smears are ■if no value in infections of the throat or
genitalia* where Streptococci may form part of the
resident flora.
For cultures, swabs should be collected under
vision fro in the affected she and either plated
immediately or sent tci Lhe laboratory in Pi kids
medium (blood agar containing 1 in 1*000,000
crystal violet and 1 in 16,0^) sodium stride). The
Specimen is pin Led cm blood agar and incubated at
37 dC anaerobically or under 5-L 0% C O ,, as
hemolysis develops better under these condition 5 .
-Sheep blood agar is recommended for primary
isolation because it is inhibitory for Haemophilus
btem oiytian , colonies of which may be r mi fused
with those of hemolytic streptococci, l [cmolytic
strep tococci are grouped by rhe Lance fie id
technique. The fluorescent antibody technique has
been employ ed for the tipid identification of group
A streptococci. A convenient method for the
identification of 5(7; pyogenes !S based OHMiTtcd?
observation th.it they an; more sensitivc to bacitradn
than other streptococci- A filter paper disc dipped
Throat or skin
Sot Hjcccbbuit
Pyodenmd types J?, 53-55, ?L> 61
and pharyngitis strains 1 and 12
Modeia te
Lowered
S ot known
Absent
Snt indicated
Sponrioeous resolution
Good
in a solution of bacitracin [1 unit/ ml) is applied on
the surface of an inoculated blood agar. After
incubation, a wide zone of inhibition is scon with
Str. pyogenes bur not with other streptococci.
Typing of7 Str. pyogenes is required only for
epidemiological purposes. If required, this may be
done by precipitation or agglutination.
Rapid diagnostic test kit* for the detection of
Streptococcal group A antigen from throat swabs
arc available commercially, "I he tests can be
completed in 1-4 hours and ore nearly as specific
as cultures* though less sensitive.
fn rheumatic fever and glomerulonephritis, a
retrospective diagnosis o f streptococcal infection
[iiay be established by demonstrating high levels
uf antibody to streptococcal toxins. The usual test
done is antistreptolysin O titration. A SO titles
h ighc r rh,i n 200 arc indicar iwe of prior streptococcal
infection. High levels are usually found in acute
rheurtU-tic fever hut in glomerulonephritis, titles
arc often low, Anti deoxyribonuclease B (anti-
1 >NAase fi) estimation .is also commonly employed.
Titles higher than 300 are taken as significant. Axiti-
DNAase fl and ajitihyoluronidfH tests, ate very
useful fur tlie retrospective diagnosis of streptococcal
pyoderma, for which ASO i* of much less value.
T h e streptozym e test, a passive slide
hemagglutination test using erythrocytes sensitised
with a crude preparation of extracellular antigens
C o p y rig h te d m aterial
of streptococci, is a convenient* sensitive and specific
screening test. Lt becomes positive after nearly all
types (if streptococcal infections, whether of the
throat or the skin.
The indication for prophylaxis in
streptococcal infection Is only in the prevention of
rheumatic fever- This achieved hy a long-term
admin ist] a [ion o f penicillin, in children who have
developed early signs o f rheumatic fever, This
prevents streptococcal reinfection and further
damage to the heart. Antibiotic prophylaxis is not
useful for glomerulonephritis as this complication
follows a single streptococcal infection, and
reinfections do not occur.
All beta hem olytic group A
streptococci are sensitive to penicillin C , and most
are sensitive to erythromycin. In patients allergic
td ]K‘nicellin, eTythromwin or cephalexin may he
used. Strains resists nr to erythromycin have been
reported. Tetracyliruea and sulphonamides are not
recommended. Antimicrobial drugs have mi effect
on established glomerulonephritis and rheumatic
fever.
Iks ides S’tr, pyogenes, streptococci belonging to
groups B, C, OhF, G and rarely H, K . O and R
may also cause human infections.
D ata from Streptococcal Reference Laboratories
in India (Lady Hardinge Medical College, New
Delhi; Christian Medical College,, Vellore) showed
that while approximately 45 per cent o f hemolytic
streptococcal isolates rested belong to group A, 1 0 -
15 per cent belong to groups B and C each* about
25 per cent to group fJ and 5 per cent to group F!
These am important pathogens of cattle, producing
bovine mastitis {Sir, agahetias). From the l% 0s.
Group G streptococcus b.i- assumed great clinical
importance as rhe single most common cause of
neonatal meningitis. in the West. Infection in the
newborn is classified as the early onset type
occurring with in a week of birth, and the late onset
type developing between the second and twelfth
weeks of life. The mure common early unset Type
presents as septicemia, m eningitis or pneumonia,
and is often fetal. Infection is acquired from the
maternal vagina din ing birth. Jn the late onset type,
Infection is m ore often obtained from the
environment. Other Group B infections in neonates
include arthritis, osteomyelitis, conjunctivitis*
respiratory infections, peritonitis, omphalitis and
endocarditis. Group B Streptococci may also cause
adult infections, including puerperal sepsis and
pneumonia.
Their ability to hydrolyse hippurate acts as a
presumptive identification method. They may be
identified by the C A M P reaction (Christie, Atkins
and Munch- Peterson), which can be dc u ions traced
as an accentuated zone of hem olysis when $tr.
agahctiiE is inocuktcd perpendicular to it streak
of Staph, s u m s grown, on hhnui agUr. Occasional
strains arc bacitracin sensitive. Human pathogenic
Group R strains possess a polysaccharide capsule
which appears to confer virulence. Nine capsular
serotypes have been identified, antibodies to which
confer type specific protection,
Streptococci of this group are predominantly animal
pathogens and may he divided into four species
biochemically. Group C strains isolated from
human source:* usually belong to Strep, equisunilis
species, it can cause upper respiratory infections,
-,is well as deep infections such as endocarditis*
osteomyelitis, brain ahsecss, pneumonia and
puerperal sepsis. Strains arc often tolerant to
penicillin and serious infections may not respond
to penicillin treatment. The addition of gentamicin
is recommended in Mfious cases, lt resembles 5rr.
pyogenes in fermenting trehalose but differs in
ferinenting ribuse. It produce? streptolysin O,
streptokinase (antigen itally distinct from that
produced by Sir. pyogtnet) and other extracellular
substances. Sir. eqaiaim itis is the source o f
C o p y rig h te d m aterial
212 4 Tyxlbce-k of ^!Cr3D blogy
streptokinase used for thrnmbotytic therapy in
patients.
GROUP F Enterococci typically appear as pairs of oval cocci,,
These grew poorly on blood agar unless incubated
under CO.,.
i Thev* have been called the "minute
strep tococci'. T hey are som etimes found in
suppurative lesions. One member of tiiis group is
Sfrrptoivcus MG which Is an alpha lytic strain
isolated from cases o f primary atypical pneuiftom. l .
Demonstration of agglu ri ni us to Streptococcus MG
in sera o f patients had been used as a diagnostic
test tor primarv atypical pneumonia.
G roup G
These are commensals in the throats o f human
beings, monkey* nr dogs. '] hey may occasionally
cause tonsillitis, endocarditis and urinary Infections
in human brings.
Groups H and K sometimes cau^e infective
endocarditis.
Group O is isolated mainly from the healthy
human throat and may cause acute tonsillitis and
endocarditis.
Group R strums are natural pathogens of pigs.
They have beer reported from occasional cases of
meningitis, septicemia and respiratory infection in
persons in co n tact with infected pigs or
contaminated meat.
G r o u p LJ S t r e p t o c o c o i
These can be classitied into twu group*: (1) the
en terococcus group fenterococ-.'i or fecal
streptococci}' which have been reclassified as a
separate genus called Enterococcus and containing
different species for example, E, facrails, E, fx riv m r
E. dunns; and (2) the nonenterecoccaf group, for
example, Str. ko\ Str. eyujnus.
The enterococci possess several distinctive
features separating them from streptococci. These
include their ability tn grow in the presence of
40 per cent bile, 6.5 per cent sodium chloride, at
pH 9.6., at 45 and in 0-1 per cent methylene
^
blue milk. On MacConkey s medium thev produce
tiny deep ]iiuk colonics. They are relatively heat
resistant, surviving 60 "C for 30 minutes.
the cells in a pair arranged at an angle to each
other [Fig. 23.4). They are usually nonhemolytic,
though some strains may show alpha or beta
hemolysis.
The identification o f enterococcus species is
made on biochemical grounds. E. f.ic.v.djj> is the
enterococcus most often Isolated from human
sources. £ liecalrs can be identified by its ability
to ferment mannitol, sucrose, sorbitol and aesvulin,
and to grow on tellurite blood agar producing black
colonics.
Enterococci are present in the intestine, genital
tract and saliva. They ;lic frequently isolated from
cases of urinary tract infection and wound infection.
They may also cause endocardium infection of the
bi Iiaiy tract, sept icon iia, and intraabdomind abscess
complicating diverticulitis and peritonitis. Strains
resistant to penicillin and other antibiotics occur
fre quently, so it is essential to perform antibiotic
sensitivity for proper therapy.
Nonenterococcal species of group D (Str. bovis,
Str. pejumus) are generally susceptible to penicillin
and are inhibited by 6.5 per cent sodium chloride
or bile. They may cause urinary infection or
endocaiditi s rarely.
T he V jr iu a n s G roup
This group, formerly called StrepTococcus vindan*,
is a cniscclkny o f streptococci normally resident in
the mouth and upper respiratory tract, and typically
producing greening (alpha lysis] on blood agai^-
hence the name vindans,
Some of them may be notily tic. They cannot be
categorized under the Lancdicld antigen, ic groups.
HoweverTbased on sugar fermentation, cell wall
composition and production of dectram and levans,
they have been classified into many species, for
example, Sir. min's, Sir mufanfr, Str. sativstrius, Str,
C o p y rig h te d m aterial
■j i *

C o p y rig h te d m aterial
214 4 ToUtw ok ol Microbiology »

r ■+!

f ir ': j..

m
MHiMi fn’ m
mfc''
r in''v tw
’-ivvnnqp
’-in ’J:
tafpvpp inHtnvi A llm tac riBirbrant showing V ctinia pooka. Note

C o p y rig h te d m aterial

an an(pa, or In short chains.
They arc ordinarily nonpathogcnic bur can on
occasion cause disease. In persons with preexisting-
cardiac lesions, they may cause bacterial
endocarditis-, Srr. s a n g u is being m ost often
responsible. Following tooth extraction 01 other
dental procedures, they cause transient bacteremia
and get implanted on damaged or prosthetic valves
or in ;t congenitally diseased heart, and grow to
fufin vegetations. Prophylactic antibiotic covet is
advisable in such persons before tooth extraction
nr similar procedures. While viridau streptococci
are generally penicillin sensitive, some strain* may
be resistant. It is therefore essential that in
endocarditis, the causative strain is isolated and Its
antibiotic sensitivity determined so that appropriate
antibiotics in adequate bactericidal concentration
can be employed for treatment.
Sfr. .'nutans (so c a lled because it assumes :l
bacillary form in acid environments! is important
]r rho causation of dcnral caries. It breahi down
dietary sucrose, producing add and a tough
adhesive dextran. 'lbc acid damages dentine and
the dextrans bind together food debris, epithelial
cell*, lsjulu.j and bacteria to lurm denial plaques,
which lead to caries. Experimental caries in
monkeys has been prevented by a Str. mutans
vaccine, but its extention to human use is fraught
with problems.

BeiKhtoji D et al, 1991, A scheme fcr identification ofiviridams streptococci. J M ed Microbiol 35 ;367l
Bisrw AL-1Wl. C>n*ip A JftrtptiKwaJ infections md acute rheumatic fever, Yew E n g J Med 325:7113.
Bisno AL and DL Stevens 1996. Streptococcal infections of slcin and soft tissues. .Yew Engl} Med 334:240.
dnirlcb 13 and H Liraen 1936. Suepioctkical puerperal sepsis Mid obstetric iiLfciiiirms: A historical perspective. C.’j J.-:
AifjLTTJiiijLjj' Rev 2 31 s.
Moellering RC. 1992. Emergence of Enterococcus as a significant pathogen- Ciin intcci Dts 14:1173.

C o p y rig h te d m aterial
 Pneumococcus
[Dipiocaccus pneumoniae: $tr> pneumoniae)
P [i e: lim o tp i; l:Li k, a G ram p ositive larii;eo]at l:
diplococcus, formerly classified as Diplococcus
pneurnoiniae, has been reclassified as Str.
pneumoniae because of its generic relatedness to
streptococcus, Pneumococcus differs from other
S tre p to c o c c i ch iefly in its m orp h olog y, bile
solubility, optochin senacivity and possession of a
specific polysaccharide capsule. Pneumococci are
normal inhabitants o f the human upper respiratory
tract, They are the single most prevalent bacterial
agent in pneumonia and in otitis media in children.
They can also cause sinusitis, bronchitis, infections
bacteremia, meningitis and other infections.
Pneumococci were First noticed itl ]SSl by
Pinteur and Sternberg independently. They
produced a tarsi septicemia in rabbits by inoculating
human «alii a and isolated pneumoCotd from the
hlond ol the animals. But the relationship between
Fig, 24.1 Pneumococci in pus
pneumococci and pneumonia was established only
in i m .
Pneumococci are typically small
fl pm), slighdy elongated cocci, with one end broad
or rounded and rhe other pointed, presenting a fame
shaped or lanceolate appearance. They occur in
pairs (dLplococci), with the broad ends in
apposition, the long axis of the cnccus parallel to
the line joining the two cocci in a pair. They are
capsulafed, the capsule enclosing each pair. The
capsules are best seen in material taken directly from
exudates and may be lost or repeated cultivation.
In Culture, the typical morphology may not be
apparent and the CoCci are more rounded, tending
to occur in short chains. They are nonmotilc and
nonsporing.
They are readily stained with aniline dyes and
arc Gram positive. The capsule may be
demonstrated as a clear halo in Indian ink
preparations nr may be stained directly by special
techniques.
Pneumococci have
complex growth requirements and grow only in
enriched media. They are aerobes and facultative
anaerobes, the optimum temperature being 37 *C
(range 25-42 DC ) and pJM 7.8 (range 6.5-8.31.
Growth is improved by 5—10% CO,.
On bland agar, after incubation for 18 hours,
the colonics arc small (0.5-1 mm), dome shaped
and glistening, with an area of green discoloration
(alpha hemolysis) around them, resembling
colonies of Sir. viridoiw. On further incubation the
colonics become flat with raised edges and. central
C o p y rig h te d m aterial
umbonatioru so that concentric rings arc seen on
the surface when viewed from above (draughtsman
or carrom coin appearance). Some strains that
develop abundant capsular material (types 3 and 7)
form large mucoid colonies.
Under anaerobic conditions, colonies on blood
agar arc surrounded by a zone of beta hemolysis
due to oxygen labile hemolysin Or In liquid media
such as glucose broth, growth occurs as uniform
rurhidity- The cocci readily undergo autolysis in
cultures due to the activity of intracellular enzymes,
Antdysis is enhanced by bik salts, sodium lauTyl
sulphate and othcT surface active agents. Heat killed
cultures do not undergo autolysis.
Pneumococci ferment
several sugars, forming acid only Fermentation is
tested in Hiss's scrum water or serum agar slopes.
Fermentation of inulin by pneumococci is a useful
test for different:ating them from streptococci at
the latter do not ferment it.
Pheumococri are bUe soluble. If a few drops of
10% sodium dcoxycholate solution are added te>
1 ml ufan overnight broth culture, the culture dears
dice I d tine lysis o f die cO^td- Alternative! v, if a loOpful
of 10% deoxychoEatc solution is placed on a
pneumococcus colony on blood agar the colony
lyses within a few minutes. fSile solubility is a
constant property of pneumococci and hence is of
diagnostic importance. Tlie bale solubiliiy rest is
bused on the presence in the pneumococci of an
autolyric amidase that cleaves the bond between
alanine and [imratnic acid in the pepridoglyraii. Hie
amidasc is activated by surface active agents such
as. bile or bile (nitta, resulting m lysis of the
organisms. The test should be carried out at neutral
pH using dccncychnlate and live young cells in saline
suspension.
Pneumococci arc catalase and oxidase negative,
Pneumococci are delicate organisms
and are readily destroyed by heat (thermal death
point 52 °C for IS minutes) and antiseptics. In
cultures, they die on prolonged incubation, perhaps
due to an accumulation, of toxic peroxides, breams
may be maintained on semisolid blood agar or by
lyophilisation.
They are sensitive to most antibiotics, beta
lactams being the drugs of choice. Almost alt strains
were sensitive to (1.05 mg penicillin till 1967, when
resistant strains began to appear.The resistance may
be intermediate (MIC 1 pg) or high (2 |ig or more)
aild due to mutation or gene transfer. The mode oi
resistance is not production of beta lactamase, but
alteration in the penicillin binding proteins on the
bacterial surface. Such strains are also resistant to
multiple drugs. A drag resistant Strep, pneumoniae
(DRSP) strain originating in Spain has spread to
most parts of the world posing problems in
tie ament.
The sensitivity of pneunnOCocd to Optochin
(ethyl hydiocuprein) 1 /5 0 0 ,0 0 0 is useful in
dilFerenriiatuig them from sneptorarn. When a disc
impregnated with oprochln is applied on a plate uf
blood agar inoculated with pneumococci, a wide
zone o f inhibition appears on iitcubxtion.
The most important
antigen of the pneumococcus is che type specific
capsular polysaccharide. As this polysaccharide
diffuses into the culture medium or infective
exudates and tissues, it is also called the 'specific
soluble substance' (SbS). Pneumococci arc
C o p y rig h te d m aterial
classified intEj lypes based on the antigenic nature
of the capsular polysaccharide. Pneumococci
Isolated from lobar pneumonia were originally
classified inm three types, I, II and 111, and a
heterogeneous group JV, Members of group IV
were Later classified into types, and now more than
90 different serotypes are recognised, named 1 ,2 ,
3, and so on.
Typing may be carried out by (1) agglutination
of the cocci with the type specific antiserum;
(2) pneci pitatinn of the SS S with the specific serum;
or (3) by the capsule swelling reaction described
hy Neufeld (1902). In the capsule swelling or
Lquellung4 reaction (quellung = swelling), a
suspension of pneumococci is mined on a slide with
a drop of the type specific antiserum and a loopful
of methylene blue solution. In the presence of the
homologous antiserum, the Capsule becomes
apparently swollen, sharply delineated and refractile.
The quellung test can he June directly with spurmu
from acute pneumonia cases. It used ro be a routine
bedside procedure in the past when the specific
antiserum was used for the treatment ofpneu mom a.
The antigenicity of the capsular polysaccharide
varies in different species. It is antigenic in human
beings and rabbits. But in mice, large doses
(500 fig) induce no immunological response
(immunological paralysis), while small doses
(0.5 |uLg) are antigenic.
Pneumococci Contain other antigens also—
a nudenproteln deep inside the cell and a somatic
'C’ carbohydrate antigen, both of which arc species
specific.
An ah ntutua L protein (beta globulin) that
precipitates with the somatic 'C antigen of
pneumococci, appears in the acute phase sera of
esses of pneumonia but disappears during
convalescence. El also occurs in some other
pathological conditions. Ihis is known as the ‘C -
reactive protein' (CRP). Its apparent antibody-like
relation to the hC ’ antigen of pneumococcus is only
fortuitous. It is not an antibody produced as a result
of pneumococcal infection. It is an 'acute phase1
substance, produced in hepatocytes. Its production
is stimulated by bacterial infections, inflammation,
malignancies and tissue destruction. Tt disappears
when the inflammatory j reactions subside. CRP is
used as an Index of response to treatment in
rheumatic fever and certain other conditions. CRP
tenting, by passive agglutination using latex particles
coated with anti-C R F antibody is a routine
diagnostic procedure.
On repeated subculture, pneumococci
undergo a smooth-to-rough ($-R ) variation. In the
R form, the colonies are rough and the cocci are
noncapsulated, autoagglutinable and avirulent. R
forms arise a-s spontaneous mutants and outgrow
the parental 5 forms in artificial culture; in tissues,
such R mutants are eliminated by phagocytosis.
Rough pneumococci derived from capsukted
C o p y rig h te d m aterial
 * Pneum ococcus ►
cells of one serotype may be made to produce
capsule-5 of the same or different serotypes,. on
treatment with DNA from the respective serotypes
of pneumococci. Th i5 Cransfbrmailion, which may
be demonstrated in vivo or in vitro, was iliscovcrcd
by Griffith (1928) and is of considerable historical
interest as the first demonstration of generic
exchange of information in bacteria.
T o x in s and u tlic r v iru le n c e f a c to r s :
PneumtHiDCr: produce an oxygen labile hemulysm
and a Icucocidin but these arc weak and make no
contribution to virulence. The virulence of
pneumococci depends on its capsule and the
production of a to^in called pneumnlysin. The
capsular polysaccharide, because of its acidic and
hydrophilic properics, protects the cocci from
phagocytosis. Capsulated pneumococci are not
phagocytosed efficiently in fluid media or exudates.
1 hey are however, Susceptible to ‘surface
phagocytosis', heing engulfed against a firm surface,
such as fibrin clot or epithelium.
The enhanced viniLence of type .1 pneumococcus
is due to the abundance of its capsular matcrial-
Noncapsufatcd strains are avirulcnt. The antibody
to the capsular polysaccharide affords protection
against infection.
Pneumolysin, a membrane damaging toxin
produced by pneumococci has cytotoxic and
complement activating properties and so may be a
virulence factor It is immunogenic- Pncumolysin
negative mutants show reduced virulence In
experimental animals. Pneumococcal autoly>ins, by
releasing bacterial components in infected tissues
may also contribute to virulence.
]*atInigenicitv: Experi mentally, fatal i nfection
can be produced in mice or rabbits by intraperitoncal
inoculation of pneumococci. Death occurs m 1-3
days, and pneumococci can be demon -.Rated in large
numbers in the peritoneal exudate and heart blood.
Phcumococci colonize the human nasopharynx
and may cause infection of the middle ear, paranasal
sinuses and respiratory tract by direct spread.
Infection of the meninges can also occur, by
219
contiguity or through blood. Pneumococcal
bacteremi.i. m.LV also lead to d^tant infections as in
the heart, peritoneum or joints. Infection is
commonly endogenous, but -exogenous infection may
also occur, especially wirh highly virulent strains.
The commonest pneumococcal infections are
otitis media and sinusii is. Prior respiratoiy infection
or allergy causing congestion and blockage
predispose to these conditions. Serotypes 6 ,1 4 ,]9E
and 231 are Commonlv if
encountered iil these
conditions, in the West.
Pneumococci are one of the most common
bactena causing pneumonia, both lobar and
bronchopneumonia. They also cause acute
tracheobronchitis and empyema.
As pi rat inn of nasopharyngeal secretions
containing pneumococci into the lower respiratory
tract is a common event and may occur even in
sleep. Normal muousal defence imccham-Hins nuoh
as entrapment, expulsion and the cough reflex, aided
by the ci 11;uy escalator eflecr prevent estabh shment
o f infection. When the normal defences are
compromised by viral infection, anesthesia, chilling
or other factors, pneumococci nultiply, penetrate
the bronchi.il mucosa and spread through the lung
along peribronchial tissues and lymphatics.
Bacteremia is common during the early stage of
lobar pneumonia. Toxemia is due to the diffusion
of the capsular polysaccharide into the blood and
rissueS- The fall of temperature by crisis and relief
of symptoms coinhie with the neutralisation of
the SSS by anti capsular antibodies.
In adults, types i-ft ate responsible for about
75 per cent of cases of pneumococcal pneumonia
and for more than 50 per cent of all fatalities due
Id pneumococcal bacteremia. In children, types 6,
14, 19 and 23 ate frequent causes.
Bronchopneumonia is almost always a secondary'
infection. This may be caused by any serotype of
pneumococcus. The damage to the respiratory
epithelium and excessive bronchial secretions
caused by the primary infection Facilitate the
iinvasion of pneumococci along the bronchial tree.
C o p y rig h te d m aterial
Bronchopneumonia is frequently a terminal event
m agLid and debilitated patients.
Ihiviaiuococci Lire commonly associated with die
acute exacerbations in chronic brontrhi ti*. rJ"be
copious respiratory sec rcrious in chmnic bronchitis
aid pneumococcal invasion Another bacterium
commonly associated with this condition is
Haemophilus influenzae.
M eningitis is the most serLou* of poeumocoocal
infectious, ]r is usually secondary to o th e r
pneumococcal infections such as pneumonia, otitis
media, sinusitis Or conjunctivitis but m 1 proportion
of cases, other foci of infection may not be
demonstrable. Pneumococcal meningitis occurs at
*11 ages. Untreated eases are almost invariably fata].
Ever with antibiotic therapy, the case fatality rate
is about 2 ? pet ee iU.
Pneumococci may also produce suppurative
lesion? in other part* of the body - eingu'ciiu,
pericarditis odds media, sinusitis, conjunctivitis,
suppurative arthritis and peritonitis, usually as
complications of pneumonia.
N'atura) infection with
pneumococci has been reported in some species of
animals such as guinea pigs but they hive little
relation to human disease. The source of human
infection is the respiratory tract of carriers and less
often, of patients. Pneumococci occur in the throat
of approximately half The population sampled at
any one time: They are transmitted from one t<i
another by fingers or by inhibition ofcontnniiiLaleil
droplets or droplet nuclei. Dissemination is
facilitated bv crowding.
Infeciion usually leads on It to pharyngeal
carriage. Disease results only when the host
resistance is lowered by contributory factors such
as respiratory viral infections, pulmonary
congestion, stress, [lulnuiricion, unmunodeficiency
or alcoholism- Splenectomy and sickle cell disease
arc important predisposing conditions
Pneumococcal serotypes vary gre.itlv in
virulence. I he case fatality rates of pneumonia
may vary according tQ The virulence of the infecting
serotype. Type 3 is rhe most virulent.
T-nbar pneumonia is usually a sporadic disease
hut epi demies mav occur an Long closed communities
as in army camps- The incidence of
bronchopneumonia increases when an epidemic of
influenza or ochtiT viral infection of tile respiratory
tract occurs. Cases are more common in winter and
Jfleet the tvyo extreme age groups m om often.
The clinical diagnosis
yt pneumonia is easy but ns the disease may be
caused by several different microorganisms,
etiological diagnosis should be made by laboratory
tests-This is of great importance in treatment-
In the acute phase of lobar pneumonia, the
rusty sputum contains pneumococci in large
numbers, with hardly any other kind of bacterium.
E’hev may he demonstrated bv Gram stain. In the
promtibiutit era, direct seretyping ol pneumococci
in wet films of sputum by the qucllung test was a
murine bedside test because success of treatment
depended on administering the specific antiserum.
In Iatcr stages nf the disease, pneumococci arc less
abundant.
The sputum l niter homogenisation if necessary,
is iiumutated or blood agar platus and incubated :it
37 °C under 5 -1 0 per cent C O r Growth occurs
after overnight incubation. Where sputum is rot
available, as in infants, serum-coated laryngeal swabs
may he used for culture. Isolation front respiratory
secretions is facilitated bv using blood agar
coilt.iming gentamicin 5 pg/ml.
From specimens where pneumococci are
expected to be scanty isolation nury be obtained by
iittrapcritoneal inoculation in mice, even if cultures
are negative. Inoculated mice die in 1-3 days, and
pneumococci may be demonstrated in the perironeal
exudate and heart blood. The test may be negative
with occasional strains that mLsvi indent tor mice
(type 14 strains).
In the acute stage (d pneumonia, the orj^nism
mav he obtained from blood culture in glucose
C o p y rig h te d m aterial
 Nlorp h u logy CapeuLred, lanceolate Noneapwlared. oval
diplocoed aa round cells in chains
Quellung rest Positive Negative
Colonics Initially -dome-shaped, Dunie-shaped
laterMnaughtsman' colonies
Growth in liquid media Uniform turbidity Granular turbidity,
powdery deposit
Bile solubility Invariably positive Invariably negative
1iiu Llcl ferm entation Positive Negative
Oprochio wnsitsvlcy Positive Negative
Intrapcritnnejl inocularicm FataJ infection Nonpaihogenic
in mice

broth. Isolation of pneumococci from blond
indicates bad prognosis.
In acute otitis media pneumococci may be
demonstrated in the fluid aspirated from the middle
car
In ease of meningitis, presumptive diagnosis
may be made Errjtrt Gtanl -Stained films ofCSF. Gram
positive diplococci can be seen berth inside the
polymorph-s and e * t rac e l lul a r ly. IJiagnosih i&
confirmed by culture. In cases which are negative
bv culture, it may be possible to establish the
diagnosis by demonstrating the SSS in CSF by
precipitation with antisera.
Capsular polysaccharide can be demonstrated
in the blood, urine and cerebrospinal fluid by
i-ounterimmunoelectrOphoresis. Antibodies can be
demonstrated by agglutination, precipitation, mouse
protection tests and bactericidal tests with whole
blood. Indirect hemagglutination, indirect FA test
and radioimmunoassay have been employed.
Immunity is type specific and
associated with antibody to the capsular
polysaccharide. The existence of some 90 serorypes
makes a complete polyvalent vaccine impracticable,
A polyvalent polysaccharide vaccine representing
the capsular antigens of 23 most prevalent serotypes
has been stated to give 80-90 per cent protection,
I[ is nut meant fur general uw, but only in persons
at enhanced risk o f pneumococcal infection such
as those will] absent Or dysfunctional spleen, sickle
cell disease, cocliac disease, chronic renal, lung,
lie art and liver disease;-, diabetes nielli rus and
immunodeficiencies including H IV infection. It is
not recommended in children under two years of
age and those with EymphoreticuJar malignancies
and immunosuppressive therapy.
The antibiotic of choice is parenteral
penicillin in serious cases anti amuaycilbn in milder
ones, provided the infecting strain is penicillin sensirivt-
Many penicillin resistant strains are also resistant to
other antibiotics like erythromycin and tetracycline.
A third generation cephalosporin is indicated in such
cases. Vancomycin is to be reserved for life threatening
illnesses with highly reidistart strains.

American Academy1of Pediatry 1997. Therapy for children with invasive pneumocoocial infection, ftdii[fries 9l£299.
Jacoba MR and PC AppLebaum 1995. Antibiotic resistant pgaenmucoed, Rev Mid AficmAvW 6;77.
Tuomanen BJ et si. 1995. Pathn^cnc-H;; g>n£,u:rKH::K.]t:a] iri.fecti.im. ,Vew f '- n g l ] \ic ti J32:12Etk
Pepvi MB- JOOl.Th* renaissance of c-reactive protein. BMJ. 322 : 4^
Quie PC ci al. 19BI. Symposium on the Pneumococcus, Rer fntict Di* Srl^S-

C o p y righted m aterial
 Neisseria

The genus Neisseria consists of Gram negative

aerobic nonsporufating, nonmulile, uJiidase positive
coed typically arranged in pairs (diplococci),
Besides the two important pathogens, j\k
meningitidis and jV . ^onorr/ioeje, the genus
COntdrs mjipy other species such as jV k t i f l l i d
that occur m commensals in the mouth or the upper
respiratory nan.

(Meningococcus;. Diplocitccuu mtraCtlluJtfio

meningitidis) Meningococcus was- first descnbfd
and isolated in 1SS7 by Weiehselbaum from the
spina] fluid of a patient.
N .m eningitidis causes meningococcal
meningitis [formerly also known as cerebrospinal
fever) which may occur sporadically as localised
outbreaks or as epidemics, and also septicemia.
■ Meningococci are Gram negative
oval or spherical cocci 0 .6 -0 8 pm in size,
typically arranged in pairs, with the adjacent sides
flattened (Kigr 2 5 .1 ). "1110 long axis of the coccus is
at right angles to a line joining the two cocci in
a pair. C o n s id e r a te variations o ccu r in size,
shape and staining properties, especially in older
cultures, due Co autolysis. In Smears hum lesions,
the cocci arc more regular and generally
intracellular, They are nonmotile, Most fresh
isolates are capsutated.
...... / Meningococci
have exacting growth requirements and do not grow
on ordinary media, Growth occurs on media
enriched with bJood, serum or ascitic fluid, which
promote growth bv neutralising certain inhibiting
substancesin' lnne media rather rl i!- prnviding
additional nutritional needs.
They are strict aerobes, no growrf occurring
anaerobe-ally.The optimum ten 1-: i ,itc: rr for growth
is 35-36 PC, No growth takes place below 30 *C.
Optimum pH is 7.4-7.6. <rrdwtli is facilitated by
5 -1 0 per cent CO , and high l untidily
On solid media, after int ibatkm for 24 hours,
the colonies are small (about 1 iffln in diameter)
translucent, round, convex, bluish grey, with a
smooth glistening surface and ■■ ii entire edges.
The colonies are typically lemiu ■1 r in shape,
butyrous in c o n s i s t e n c y and easily e n i u l s i f L l h l e .
Weak hemolysis occurs or blood agar. Smooth ami

C o p y rig h te d m aterial
 4 Ne'sseMa ►
rough Types of colonies are found. Growth is poor
in Iiqu i3 med is, prndu ci :ig a granular turbid itv wi th
little or no surface growth.
Blood agar, chocolate agar and Muelle^Hinton
starch casein hydrolysate agar are the media
commonly used for culturing meningococci.
Modified Thayer-M artin {with vancomycin,
colistir and nyntati n) it a useful 5elcchve medium.
ftiocheitiioflJ reuti^HiBi LJ'hcy arc catalase and
oxidase positive. The prompt oxidase reaction helps
the identification of neisseria (both meningococcus
and gonococcus) in mused cultures. When freshly
prepared 1% solution of oxidase reagent (tetrameihyl
paraphenylene diamine hydrochloride) is poured
on the culture media, the neisseria colonies turn
deep purple. Subcultures should be made
immedlately, as the organism dies on prolonged
exposure to the reagents The test may also be
performed by rubbing a little of the growth with a
loop on a strip of filter paper moistened with the
oxidase reagent (Kovac's method). A deep purple
colour appears immediately.
Indole and hydrogen sulphide are not produced
and nitrates axe not reduced- Glucose and maltose
are utilised, but not sucrose or lacrosc, producing
acid but no gas (gonococci acidify glucose but not
m altose). And form al ion hv ne isserii ;lc is weak,
:v LngoocdaEi ve and therefore best tested on peptone
scrum agar slopes containing the sugar and
indicator.
A nlicon ic p ro p erties and c ln s s iflu at ion:
M eningococci are capsulated, unlike other
nelsseriae. Based on their capsular pulysaccande
antigens, meningococci arc classified into at least
13 senognoups, of which Groups A, B and C arc
the most important. Group A is usually associated
with epidemics and Group C mostly with localised
outbreaks, while Group R causes both epidemics
and outbreaks. Groups 2 9 -E , W -135 and \ also
frequently cause meningitis-. Any semigroup may
colonise the nasopharynx, but these six groups
account for the large majority of meningitis.
Serogmups are further classified into serotypes and
223
subtypes based on outer membrane proteins and
polysaccharides.
R eb istan ce: Meningococci arc very delicate
organisms, being highly susceptible to heat,
dedication, alterations in pH and to di-lnlecEants.
They were uniformly sensitine to penicillin and
other antibiotics, but resistant strains have emerged
and become common m many uri'.is.
P ath ogen icity: Cerebrospinal meningitis and
menuigoooecal septicemici are the two mam types
of meningococcal disease. Meningococci are strict
human parasites inhabiting the naso ph arynx.
Infection is usually asymptomatic. In some, local
inflammation ensues, with rhinitis and pharyngitis.
Dissemination occurs only in a small proportion.
The manner in which the cocci spread from
the nasopharynx tn the meninges may he directly
along the perineural sheath of the olfactory nerve,
through the cribriform plate to the subarachnoid
space, or more probably, through the bloodstieam.
In certain cases the -.itc of entry of the
meningococcus may be the conjunctiva. Cases of
meningococcal purulent conjunctive is occur. On
reaching the central nervous system, a suppurative
lesion of the meninges is set up, involving the surface
of the spinal cord as well as the base and cortex of
the brain. The cocci are invariably found m the
spinal fluid, both free and within the leucocytes.
Case fatality l* variable but in untreated cases may
be as high as SO per cent. Survivors may have
sequelae such as blindness and deafness. Some cases
develop chronic or recurrent meningitis
M eiungucotcem u presents as acute fever with
chills, maliLLse and prostration. Typically a petechial
rash occurs early in the disease. Meningococci may
be isolated from the petechial lesions. Metastatic
involvement uf the joints, ears, eyes, lungs and
adrenals may occur. About 10 per cent develop
pneumonia.
A few develop fulminant metiingococccmia
(formerly called W aterhouse-Friderichsen
syndrome) which is an overwhelming and usually
fatal condition, characterised by shock, disseminated
C o p y rig h te d m aterial
224 4 Tflsttbook of Microbiology t
intravascular coagulation and multisystem failure.
Rarely chronic meningococcemia may be seen.
Meningococcal disease is favoured by deficiency
of the terminal complement components (C 5-C 9).
The pathogenic agent in meningococcal disease
appears to be the endotoxin [LPS) released by
autolysis. The vascular endothelium is particularly
sensilive to the endotoxin. All maytf inflaminatoiy
cascade systems as well as cytokines and nitric oxide
are triggered and upregulated. In fulminant cases
adrenal hemorrhage and profound shock are
present.
Natural infection is limited to human beings.
[ntraspLuai inoculation of large numbers of cocci
may produce a picture of meningitis in monkeys-
Irtraperitoncal inoculation of the cocci suspended
in hog gastric mucin brings about a Mu I infection
in mice.
Epidemiology: The human lusoyluryiu is the
only reservoir of the meningococcus. Asymptomatic
nasopharyngeal carriers rarely contract the illness
hut serve to infect their contacts. Transmission is
essentially by airborne droplets or less often by
foriiTes. During intercpidcmic periods, the carrier
rate is about 5 -10 per cent. An increase in carrier
rate heralds the onset of an epidemic. During
epidemics the earner rates in closed communuiL'E
may go up to VO per cent. Meningitis is common
m children between 2 months and 5 years of age.
Epidemics usually occur in semidosed communities
living in crowded conditions, as in jail; and ships
formerly, and in army camps in recent times.
Prevalance of meningitis le highest in the
'mcningiris belt of Africa'stretching from Ethiopia
to Senegal. Frequent epidemics have occurred here.
One of the largest was in lV9h, when 150,000 cases
and 15,000 deaths were reported.
L a b o r a to r y J ingot jni-c The primary agents
causing purulent bacterial meningitis are
meningococci, pneumococci and f/flnnopfi/foj
influeniae type h. Other important causative agents
are group B streptococci, staphylococci, fxiifrif/ijff
coir and LiVrm'a mwnxyftigenes.
It is necessary to establish the specific etiology in
purulent meningitis for proper treatment. In
meningococcal meningitis, the cocci arc present in
large numbers in the spinal fluid and, in the early
stage, in the blood as well. Demonstration of
meningococci in the nasopharynx helps in the
detection of earners,
1. Ejsxmiiwrion o fC S fi The fluid will be under
pressure and turbid, with a large number of pus
cells. For bacteriological examination, if a
sufficient [|uamity is available, the C SF is
divided into three portions. One portion is
centrifuged and Gram stained smears are
prepared from the deposit. Meningococci will
he seen mainly inside polymorphs but often
cxtracellulariy also. This presumptive diagnosis
is sufficient to start antibiotic treatment. The
supernarent will contain meningococcal antigen,
which may be demonstrated by latex
agglutination or counter Immunoelectrophoresis
using meningococcal antisera. Similar testa are
also available for pneumococcus, H. influenzae
type b and Group B streptococcus antigens.
Antigen detection is particularly useful in
parti.illy treated patients in whom smear and
culture tests may be negative. The second
portion of the CSF is inoculated on blood
agar or chocolate agar plates and incubated at
35-3fi ^C. under 5 -1 0 %l CO^ Colonics appear
after IS—24 hours and may be identified by
morphology and biochemical reactions. It is
important to remember that morphologically
similar organisms such as Y, /Javcsccns. Y, /Java
and Acinetobacter may also cause purulent
meningitis occasionally. The isolated
meningococcus may be grouped, if required, by
agglurinatinn with the appropriate sera. The
third pot lion of CSF is incubated overnight*
either as ir is or aftc r add ing a n equal volume of
glucose broth and then subcultured on
chocolate agar. This method may sometimes
succeed where direct plating fails.
2. Stood culture: In meni:lgucuCcrriio and in early
C o p y rig h te d m aterial
 case? of meningitis, blood culture is often
positive- Cultures should be incubated for 4~7
days, with daily subcultures. Meningococcal
antigen? can be found in the blood in active
disease.
3. This is useful tor the
detection of carriers. Sampling should he done
without contamination with saliva, The swab
should he held in a suitable transport medium
(for example* Stuart's) till it is plated,
4. M eningococci may
sometimes- be demonstrated in petechial lesions
by microscopy and culture.
5. At autopsy specimens may be collected
from the meninges, lateral ventricles or the
surface of [lie brain and spinal cord for smear
and culture. Meningococci may die if specimens
art nor collected within twelve hours of the
death of the patient.
Retrospective evidence
of memftgQCOCCal infection may he obtained by
detection of antibodies.
7. Group specific diagnosis
of Infection fan be made by detection of
meningococcal 1>NA sequence in CSF or blood
by PCR amplifications.
Prompt treatment is essential to
ensure recovery without sequelae. Sulphonamides,
onde the mainutav, are nat used now due to
widespread resistance. Intravenous penicillin G is
the treatment of choice. Chloramphenicol is equally
effective. One of the later cephalosporins
(ceftriaxone, ceftazidime) may be used for tht
initiation of treatment before the etiology of
meningitis i* known.
After the initial course of treatment, cradicadve
therapy is to be giver with rifampicin or
ciprofloxacin to free the nasopharynx of the cocci
and prevent carrier state,
Sulphonamidea arc not effective
due to resistance. Penicillin is unable tu eradicate
the carrier state. Rifampicin or Ciprofloxacin is
recommended for chemoprophylaxis. As attack rates
ate very high in the household or close contact? of
meningococcal patients,, they should be provided
with chemoprophylaxis.
Monovalent and polyvalent vaccines arc available
containing the capsular polysaccharides ul groups
A, L\ W -135 and V, The vaccines induce good
immunity after a single dose in older children and
adults hur arc of little value in children below
3 yean. The immunity is group specific. There is
no Group B vaccine available at present.
X . g o n o r rh o e a causes the venereal disease
gonorrhea. The gonococcus was first described in
gonorrheal pus by N erase; in 1879. Burmin in 1885
cultured the coccus and proved its pathogenicifv
by inoculating human volunteers. Gonococci
resemble meningococci very closely in manv
properties.
In smears From the urethral
discharge in acute gonorrhea, the organism appears
as a diplococcus with the adjacent sides concave,
bring typically kidney-shaped. It is found
predominantly within the ptilvrtiorpEi*, some cells
containing as many as a hundred cocci-
Gonococci possess piJi on their surface. Pill
facilitate adhesion oi the cocci to mucosal tiurtaces
and promote virulence by inhibiting phagocytosis.
Pillared gonococci agglutinate human red. blood
cells hut not red cells from other mammals. The
hemagglutination is not inhibited by mannose,
Gonococci are
more difficult to grow than meningococci. They
are aerobic but may grow anaerobically also.
Growth occurs best at pH 7.3—7.6 and. at a
temperature of 35-3f> ‘C. It is essential to provide
5-10% COy They grow well on chocolate agar
and Mueller-H inton a^ar. A popular selective
medium is the Thayer-Marim medium (containing
vancomycin, colisrin and nystatin.) which inhibits
most contaminants including rtonp.itbogenic
neisseria.
Colonies arc small, round, translucent, convex
C o p y rig h te d m aterial
226 ■* Tauctbook of Micro biology
or slightly umbonatc, with finely granular surface
and lobate margins. They are soft and easily
emu] 'i tiablc.
Four types of colonies have been recognised -
T l to T4. Types 1 and 2 form small brown colonies.
The cocci - l i t j i iIi,H lll ] , auDoagglu unable and vi mlent.
Types 3 and 4 form larger, granular, nonpigmcntcd
colonies. T 3 and T4 cocci are nonpiliatcd, form
smooth suspens iims and are avi rulcnf, Fresh i*oktcs
from acute cases of gonorrhea generally form T l
and T2 colon lies. On serial subculture, they change
to T3 or T4 colonial morphology. T l and T2 types
arc also known as P - and P " , respectively, while
T3 and 1 4 are known as P\
B iDuhemical reactio n E Gonococci resemhle
mcni ngococci except in the effect on maltose-
Gonococci acidify only glucose and not maltose.
A uligun it: p r o p e r t ie s Gonococci are
antigen ually heterogeneous. ] hey ate capable of
changing their surface structures in vitro. I'hey
probably do so in vivo as well, to avoid host defence.
The surface structures include the following;
Pill which are hairlike structures several
micrometres long, act as virulence factors by
promoting attachment to host cells and inhibiting
phagocytosis. Pill are composed of repeating peptide
subunits (pilins) consisting of conserved (constant)
and variable regions. Pl'i undergo anugemc and
phase variation.
The trilaminar outer membrane of gonococci
contains many different proteins. Protein I is the
major constituent ind shows antigenic diversity,
which helps in typLrtg gonococcal strains. Protein 1
of a single strain is mdgenlcalJy constant, though
it shows considerable heterogeneity between
different strains, "llicie are two variants of protein L,
called IA and IE, Any one strain carries only either
LA or IB but not both. Using monoclonal antibodies
to protein [ epitopes, gonococci can be classified
into several scrovars, Al to 24 and Bl to 32.
Proteins I and I I I act as ligands attaching the
coccus to the host cells. Thev also form
transmembrane channels (porms) which play a role
in the exchange of molecules across the outer
membrane.
Protein I I is related to the opacity of the
gonococcal colonies and so is called the 'opacity
associated' (OPA) outer membrane protein. Strains
with the OPA protein form opaque colonies and
those lacking it transparent colonies. A strait] may
express 0 to 3 serological varieties of the OPA
protein at a rime- OPA may be responsible for
attachment to the host cells and also for the
clumping af cocci seen in urethral exudate smears.
1’hc outer membrane also contains
lipopolysacchaf.de (endotoxin) which may be
responsible for the toxicity in gonococcal infections.
Many other proteins of poorly defined rules in
pathogeniciry have been described. Both gonococci
and meningococci elaborate IgA 1 protease that
splits and inactivates IgA.
Kcsistunoc! The gonococcus is a very delicate
organism, readily killed by heat, drying and
antiseptics. It is a strict parasite and dies in 1-2
hunts in exudates outside the body.
In cultures, the coccus dies in 3—4 days but survives
in slant cultures at 35 “C if kept under sterile paraffin
oil. Cultures may bo stored for years if frozen
quickly and left at -7 0 °C.
Pathogen icityt Gonorrhea is a venereal disease
which has been known from ancient times. The
name gononhea (meaning* flow of seed) was first
employed by Galen in 130 AD.
The disease is acquired by sexual concact. The
first step in infection is adhesion of gonococci to
the urethra or other mucosal surfaces. Fill are
invoked in this adhesion. Adhesio.ii is rapid and
firm so that micturition after exposure offers no
protection against infection. The cocci penetrate
through the intercellular spaces and reach the
Subepithelial connective [issue by the third day after
infection. The incubation period is 2 -8 days. In
men, the disease starts as an acute urethritis with a
mucopurulent discharge containing gonococci in
large numbers. The infection extends along the
urethra to the prostate, seminal vesicles and
C o p y rig h te d m aterial
 * Melswria >
epididymis, Chronic urethritis may lead to stricture
form ation. T h e infection may spread to the
periurethral tissues, causing abscesses and muleipie
di-,chargmg sinuses ('wacercan perineum1).
In women, the initial infection involves the
urethra and cervix uteri, The vaginal mucosa is not
usually affected in adults because the stratified
squamous epithelium is resistant to infection by the
COCCi and also because at the acid pH o f vaginal
secretions. {Vulvovaginitis occurs in prepubertal
girls). The infection may extend to Bartho'.iifs
glands, endometrium and fallopian tubes. Pelvic
inflammatory disease and salpingitis may Lead l-h
sterility. Rarely, peritonitis may develop w ith
perihepa-"- inflam m ation CPLt-z—I lu g h -C u rtii
syndrome). Clinical disease is as a rule less severe
m women* many ofw liom may cany gonococci in
the cervix without any symptoms. Asymptomatic
carriage o f gonococci is rare in men.
Proctitis occurs in both -sexes. It may develop
by direct contiguous spread in women but in men
is usually the result o f anal sex. Conjunctivitis may
occur, usually due to auti linocuSation by the patient's
fingers. Blood invasion may occur from the pri maty
site o f infccrion and may lead to metastatic lesions
such as arthritis, ulcerative endocarditis and very
rarelv meningitis. Occasional cases o f pyemi.ii have
been reported-
A nonvcncteal Infection is gonococctil
ophthalmia in the newborn h which results from
direct infection during passage through the birth
canal. Gonococcal bacteremia leads to skin lesions,
especially hemjorT:i.Lgic papules and pustules on tlie
hands* forearm, feet and legs, and to tenosynovitis
and suppurative arthritis,, usually o f the knees, ankles
and wrists.
Gonococci ccmtaio several plasmids, Xinety-five
per cent o f the strums have a small cryptic plasmid
o f unknown function. Two other transmissible
plasmids contain genes that code for beta lactamase
wh-ch causes rcs:-■stance to penk'llin-
I'htidcjnidLnfiyL G onorrhea is an exclusively
human disease, there be mg no natural irtfetLion in
227
animals. Experimental disease may be produced hi
chimpanzees by urethral inoculation. A lethal
infection can he produced in mice by intracerebral
inoculation.
The only source o) infect ion is a human carrier
or less often a patient. The existence o f asymptomatic
carriage in women makes them a reservoir serving
to perpetuate infection among their male contacts.
The m ade nt infection is almost ekcI u :-icelv
' f venereal.
Fomiccs do not play any significant role, as the cocci
die rapidly outside the human body. T h e only
nonvenercal infection is ophthalmia neonatorum.
Once very cummun, th is has been controlled by
the practice of instilling 1% silver nitrate solution
mro the eyes of all newborn babies {Crcde'a method).
When sulphonamides, and later penicillin, were
found very effective for the treatment of gonorrhea,
it was hoped that the disease could be eradicated.
However, after a temporary decline, its incidence
has been rising steeply. In 1970, the global incidence
of new cases was estimated at 16 million, making
it one of the most common infective diseases. In
same areas, gonorrhea has reached epidemic
proportions, especially i.n adolescents and young
adults. T in- reasons lor the increase in gonococcal
infection are largely social and cultural. In the
19BDs, with the AIDS scare, there was a noticeable
decline in the incidence of gonorrhea, but thi- has
not been kept up, A higher incidence of gonorrhea
has been observed in persons belonging to blood
group B. The ba.-i> for this is not known.
L a b o ra to ry dia&noaift: In the acute stage,
diagnosis can be established readily bolt chronic
cases sometimes present j^rul difficulties. Ill acute
gonorrhea the urethral discharge contains
gonococci in large numbers. The meatus is cleaned
with a gauze soaked in saline and a sample o f the
discharge collected with a platinum loop fbr culture,
or directly on slide for smears. In women* besides
the urethral discharge, cervical swabs should also
be collected. This should hi.- done carefully, using a
speculum. High vaginal swabs arc not satXfoctory.
In chronic infections, there may not be any
C o p y rig h te d m aterial
urethral discharge. The 'morning drop' of secretion
cnay be examined or some exudate may be obtained
alter pm^tifu' massage. It may also be possible to
demonstrate gonococci in the centrifuged deposits
of urine in cases where no urethral discharge is
available.
The demo m iration of intracellular C ram
negative diplococci in stained smeara provides a
presumptive evidence of gonorrhea in men. It lias
to be emphasised that diagnosis of gonorrhea hy
smear examination is unreliable in women as some
of the normal genital flora have an essentially
similar morphology. The use of fluorescent
antibody techniques for the identification of
gonococci in smears has increased the sensitivity
and specificity of diagnosis by microscopy.
For culture, specimens should he inoculated (in
prewarmed plates, immediately on collection. J f this
lhnot possible, specimens should be collected with
charcoal impregnated swabs and sent to the
laboratory in Stuart's transport medium. In acute
gonorrhea, cultures can be obtained readily on
chocolate agar or .Muellc i—H:titon agar incubated
at .15-3h "C uniter .5-10% COj. In chronic cases,
where mixed infection is usual and in the
examination of lesions such as proctitis, however,
it is better to use a selective medium such as the
J^biyemMartin medium, The growth Is identified
by morphology and biochemical reactions.
It may not be possible to obtain gonococci in
culture from some chronic cases or from patients
with metastatic lesions such as arthritis. Serological
Tests may be of value in Such instance*. The
complement fixation test has been used with varying
degrees of success. It becomes positive only some
weeks after the infection is established and may
remain positive fur months or years after the disease
has been cured. The test may also be positive
following meningococcal infections. It is necessary
to use a polyvalent antigen because of the antigenic
heterogeneity of the gonococcal strains. The test is
not suitable for routine use. Many oilier serological
tests have been attempted, including precipitation,
passive agglutination, immunofluorescence* and
radioimmunoassay using whole-cell lysate, pilus
protein and lipopolysaccbaride antigens, However,
nn serological test has been found useful for routine
diagnostic purposes.
In 1935', when sulphonatnidcs were
introduced for treating gonorrhea, all strains were
sensitive to the drug but resistance developed
rapidly Again, when penicillin was introduced, all
strains were highly sensitive (MIC 0.005 unit/ml).
From 1957, strains with decreased susceptibility
(MIC higher than 0,1 unit/ml) became common.
As patients infected with such strains did not
respond to the usual doses of penicillin, very large
doses of penicillin, 2.4 million units were used.
From 1976, gOnOCOCCi producing |3 Lactamase
(penicillinase) have appeared, rendering penicillin
treatment ineffective. These penicillinase-producing
JV, gopoirhottt (PPNG} have spread widely.
The Centers for Disease Control and Prevention,
USA in 1993 recommended the following schedule
lor uncomplicated gonorrhea: Ceftriaxone 125 mg
single INI dose or Ciprofloxacin .500 mg (or
C o p y rig h te d m aterial
Ofloxacin 400 mg) single oml dose, plus
DoxycytJine 100 mg twice daily for 7 days of
Erythromycin 1 g single oral dose. The regunen is
costly hut work* very well against gonococci and
the frequently coexisting chlamydial infection.
Control of gonorrhea consists of
early detection of cases, contact tracing, health
education and other general measures. As even
clinical disease docs not confer any immunity,
vaccination has no place in prophylaxis.
Along with an increase in the Incidence of
gonorrhea, there has also been an increase, in recent
years, nf cases o f chronic urethritis where gonococci
cannot be demonstrated. This has been called
nongonococcal Or [lons^iecific ore [but is. In some
of these, urethritis forms part of a syndrome
consisting o f conjunctivitis and arthritis in addition
(Reiter's svndrome). Some o f these cases mav be
due 10 gonococcal infection, till: coed persisting as
L-forms and hence undetectable by routine tests.
Tbe majority o f such cases are, however, the result
of infections nf diverse etiology. The most important
o f these arc Cijfam . trachoma Of, Ureaplasma
urca^fjcum and M y n ^ b n iu hominis. Jleipes virus
and cytomegalovirus may also account for some
cases. Urethritis may also be caused by ocher
bacteria (fo r example, Gardncrclla vaginalis,
Aciaetobacter Iwaffi, i4c. cdrddcetiojtj., /ufr^j'
(C-tndidp afhii-ans), protozoa (Trichom onas
vaginalis), or even by mechanical oi chemical
irritation. As etiological diagnosis is seldom
achieved, the management of this syndrome is
difficult,
Several species of Ncisscriae inhabit the normal
ieS] hiratoty tract- The characteristie features of S >cne
of the common species ire listed in Table 25,1,
<

<

IV. mcnigitidit Round, smooth, T h ir te e n

1

shiny creamy m b g tin ic

consistency group*
l\ ' .
^ono rr/io eae Same as above, A A n tig e n ic a lly
bur sm aller and heterogenous
m ore opalescent
N. (hvescens R e s e m b le +■ 4 A n tig e n i ra lly
meningococcus but d is tin c t
p igm ented y ello w hom ogeneous
g ro u p
Ar. sicca Small, dry, opaque, * + A A A Aumogghirinahfc
w rin k le d , b rittle
N. caJUn-hairs V ariable, sm ooth * t Aulous^utinabk:
(Branhantelll and tflftd u c e n t Of
catanbalir) adherent and
opaque, not easily
e m u lrifia b le

C o p y rig h te d m aterial
23 0 * T n rttm fc of kti ^robiology »

Their pathogenic significance is uncertain though
some o f them, (for example* N , fiave&cen&y N,
cafarriiaiiV) have been reported occasionally as
h.iving caused meningitis.
.V. 7acfamjL’ah frequently isolated from the
nasopharynx is closely related to meningococci,
though it is virtually avindent. It differs from
pathogenic neisseria? in being positive in the
ONPG test for beta galactcH-idase. Nasopharyngeal
colonisariisn by jV Jactanun in young children may
be responsible for rhe presence in them of antibodies
probecrivc against mcniiigococcal infection,
Neisseria catarrhalis is now cl as tiffed at
Morixella (Branhxmciia) catarrhulis. It i* an
opportunistic pathogen capable of causing laryngiti ^
bnonchopncumonia, men irgitis, sihuf i iis and middle
ear disease.

Further Keailiaj
Britigan BE and PF Spar .mg 1965. Gonococcal infection. New Engl J Med. 1 16# :.
Cartwright K fed) 1995. Afen/r^pocTDcraJ Diseaset Chichester John Wiley.
M y p e SA j L 1?S9. iiv?i :in [Kuhnigrpi.- Ncisswfii*- ¥pp. C fo MrcmbitiJ (Svppl-lSj-
Roicntstcm NC rt nl. 2001. Men insocwcal disease!, N ew ffrtgf J M ed , 334:1376
TiinkeE AR and MW Schrld 1995. Acute bacterial meningitis. Lancet, 346:1675.

C o p y rig h te d m aterial
 Corynebacterium
Corynebacleru are Gram positive, iluuaciiJ fi£t,
nonmotile rods with irregularly stained segments,
and sometimes granules. They frequently show dub
shaped swellings and Hence the name
coiyncbactcria (from ctuyne, meaning' club). "l'hc
most important member of the genus is
C- diphthcriae, the causative agent of diphtheria.
Diphtheria has been known from ancient times.
Aretacus, the Cappadocian, in the second century,
described the Egyptian or Syriac ulcer, which most
medical historians agree can he identified us
diphtheria. The disease was first recognised as a
clinical entity by Hretonneau (1826) who called it
'diphthcrirc'. The name is derived from the tough,
leathery pseudo-membrane formed in the disease
(d/phrheros, meaning leather). The diphtheria
bacillus was firm observed and described by Klebs
(1R83) but was fust cultivated by Larefflrf (1884).
Jt is hence known as the Klcbs—1 -oeffkr bad Hus
or KLB. Loefflcr studied the effect of the bacillus
in experimental animals and concluded that the
disease was due to some diffusible product of the
bacillus. Roux and Yersin flSSS) discovered the
diphtheria exotrarin and established ils pathogenic
effect. The autitcudn was described by von Behring
(1 8 9 0 ).
The diphtheria bacillus is a slender
rod with a tendency to duhbing at one or both
ends, measuring approximately 3 -6 pm * 0.6-0.5
Jim. The bacilli arc pleomorphic. They are
nonspoting, noncapsulated and nonmotile. Cells
often show septa, and branching is infrequently
observed. They are Gram positive but tend to be
decolorised easily. Granules composed of
prilymctaphnsphate are seen in the cells. They are
mo re strongly Gram positive than [he rest of the
bacterial cell- Stained with Loefflct’s methylene
blue, the granules take up a bluish purple colour
and hence they are called me [achromatic granules.
They are also called vrJuhnor Bxbes ErrtSfjframifes.
They arc often situated nr the poles of the bacilli
and are called polar bodies. Special stains, such as
Albert’s, Neisseria and Ponders have been devised
for demonstrating the granules clearly. The bacilli
ate arranged in a characteristic fashion in smear?;.
Thcyan; usually seen in pairs-, palisades (resembling
stakes of a fence) or small groups, the bacilli being
at various angles to eath other, resembling the
letters V or L, This has beer called the Chinese
letter or cuneiiorm arrangfmcnf.This is due to the
incomplete sepn ration of the daughter cells after
binary fission (Fig. 26-1).
Growth is scanty
on ordinary media. Enrichment with blood, serum
or egg is necessary for good growth. The optimum
temperature for growth is 37 °C (range 15-40 °C)
and Optimum p ll 7.2r It is an acrp.be and a
facultative anaerobe.
The usual media employed for cultivation of
the diphtheria bacillus are Loeffler's serum slope
and tellurite blood agar. Diphtheria bacilli grow
on Loeffler’s serum slope very rapidly and colonies
can be seen in 6 -8 hours, long before other bacteria
grow. Colonies are flt fin;t Small, circular white
opaque discs hut enlarge on continued incubation
and may acquire a digrind yellow tint. Several
C o p y rig h te d m aterial

modifications of relluriie blood agar hive been
utilised* such as M cLeod’s and Hoyle’s media.
Tellurite (0,f>4%} inhibit:; the growth of mwl other
bacteria, acting as a selectivr agent. Diphtheria
bacilli nc-ducc tellurite io metallic tellurium, which
i> incorporated in the colonies giving them, a grey
or black colour. The growth of diphtheria bacilli
may he delayed on the tellurite medium and colonics
mav take two days to appear. Based on colonial
morphology on the tellurite medium and other
properties, McLeod classified diphtheria bacilli into
three types - gravis, intermedins and miris, The
names were originally proposed t" relate to the
clinical severity of the disease produced by the three
types - gravis, causing the most serious, and mills
the mildest variety, with intermedins being
responsible for disease of intermediate severity.
However, this association is not constant. The
necessity for typing an isolate in the laboratory' has
been superseded by the need to know whether the
strain is toxigenic or not. Certain biological
characteristics of these individual types have some
value.
The gravis, and intermedius types are associated
with high case fatality rates, while mitis infections
are less lethal. Paralytic complications are most
common in gravis, hemorrhagic complications in
gravis and intermedius, and obstructive lesions in
the air passage in mitis L:i fee Lions. In general, nil Us
is the predominant strain in endemic areas, while
gravis and intermedius tend to be epidemic. The
mitis type is better able than the more virulent types
in establish a commensal relationship with the
host. Wide variations have been noted in the
frequency of the different types in different places
at different times-. There is evidence to show that
the gravis And, ro a lesser extent, the intermedius;
strains are able to spread more readilv than the
mitis in populations naturally immune or artificially
immunised. Table 26.1 lists the characteristics of
the three types.
Diphtheria bacilli ferment wirb the production
of acid, (but no gas) glucose, galactose, maltose
and dextrin (hut not lactose, mannitol or sucrose).
Some strains of virulent diphtheria bacilli have been
found to ferment sucrose. It is necessary to employ
Hiss’s serum warier for nesting sugar fermentation.
Priiteolytic- activity is absent.They do not hydrolyse
urea or fnmi phosphatase.
Vincent stnai ns of diphtheria bacilli produce a very
powerful exotoxin. The pathogenic effects of the
bacillus are due to the toxin. Almost all strains of
gravis and intemuedius (about 95-95 per cent) are
tori genic", while only about BO—SS per cent of the
rniris strains arc so. The proportions vary with the
origin of the cultures tested. Strains of all three
types are invariably virulent when isolated from
acute cases, Avirulent strains arc common among
convalescents, contacts and carriers, particularly in
those with extrafiucial infection. There is
considerable variation in the amount of toxin
produced by the different strains, some strains
producing it aWndantly jnd others only poorly. But
the toxin produced by all the strains of diphtheria
C o p y rig h te d m aterial
 lorphology Usually short rod,, with
uniform staining, few or nn
granules. Sonic decree of
pleomorphism, with irregulsriv
barred, sflow^shoe and tear-
drop forms
Colony on tellurite In 18 hours, colony is l -2 mm
blood agar in size, with greyish black
centre, paler* semitranslucent
periphery and commencing
aeration of edge. 1n 2-,3 days,
3-5 mm in xi.K(;, jl.it colony
with raised dark centre and
Lrenated edge with radial
stoat iun - "daisy head" colony
Consistency of Like 'cold margarine', brink,
colonies faihvcs js .i whole on the plate,
not easily picked out or
emuliifiable
Hemolysis Variable
Growth in hroth Surface pellicle, granular
deposit* Little or no turbidity
Glycogen and iBnfh Positive
fermcnE^ison
bacilli is qualitatively similar. The strain almost
universally used for toxin production Is the 'Park
Williams S’ strairij which has been variously
itev-rrilieJ as a mil is (Topley and Wilsinl] and an
irtermedius strain (Cn.iickshank),
T h e diphtheria teaxiti is ;l protein and h*e- Ihteii
crystallised. It has a molecular weight of about
62,000, It is extremely potent and the lethal dose
for a 250 g guinea pig is- 0.0001 mg. It consists of
two fragments* A and B, of M W 24,000 and 38*000*
respectively. Both fragments arc necessary for the
toxic effect. When released by the bacteriumhthe
Long V-j.rrc.-Li forms with [.ong, curved,
duhM ends; poor pleomorphic rods with
granulation, very prominent granules
pleomorphic
18 hour colony small. Sik variable. shiny
Irmn in size, misty. Dots black. In 2-3 days,
nut enlarge in 4B hours, colonies become flat*
dull granular centre with with a central
Einwthtr, mnre elevation “polished egg"
listen ing periphery and colony
a lighter ring n ar the
edge - "rio g 'i egg" colony
Intermediate between Soft, buttery, easily
gravis and mitia emidsi liable
Nonhemolytic Usually liemolytic
Turbidity in 24 hours, Diffose turbidity with
clearing in 4S hours* -soft pellicle later
with line granular
sediment
Negative Negative
toxin is inactive hecause the active site on fragment
A is masked- Activation is pmhably accomplished
by proteases present in the cuEtuce medium and
infected tissues. All the enzymatic activity' of the
toxin is present in fragment A. Fragment B is
responsible for bin ding the toxin to the cells* The
antibudy to fragment B is. protective by preventing
the binding of the toxin to the cells. The toxin is
labile. Prolonged storage, incubation at .37 "C for
4—6 weeks, treatment with 0.2-0.4 percent formalin
or acid pH converts it to toxoid. Toxoid is toxin
that has lott its-toKieity bur nor ire antigenicity, It is
C o p y rig h te d m aterial
capable of inducing antitoxin and reacting
specifically with it.
The toxigenic icy of the diphtheria bacillus
depends on the pr esc nee in it of corynephagcs
which act as the genetic determinant
controlling toxin production. N'tinloxigenic strains
may he rendered toxigenic by infecting them with
bera phage or some other toxlargcr phage. This is
known a? lywgcnic or priag-e toniwrsif^ Tlie
toxigcnicity remains only as long as the bacillus is
Ivsogentt- When the bacillus is cured of its- phage,
as by growing it in the presence of antiphage serum,
it loses die toxigenic capacity.
Toxin production is also influenced by the
concentration of iron In the mcdffUm.The optimum
level of iron for toxin production is 0.1 nig per
litre, while a concentration of 0.5 mg per litre
inhibits the formation of toxin. The diphtheria
toxin acts by inhibiting protein synthesis.
Specifically, fragment A inhibits polypeptide chain
elongation in the presence of nicotinamide adenine
diniicJcuride hy inactivating the elongation factor,
EF-2, It has a special affinity for certain tissues
such as the myocardium, adrenals and nerve
endings.
Cultures mav remain viable fer two
or more weeks at 25-30 “C. It is readily destroyed
hy heat in ID minutes at 5K :'C and in a minute at
100 ° C It is mote resistant tn the action of light,
desiccation and freezing than most nonsporing
bacilli. Ir has been cultured From dried bits of
pseudomembrane after 14 weeks. It re maim- fully
virulent in blankets and floor dust for five weeks. It
is easily destroyed by antiseptics. It is susceptible
to penicillin, erythromycin and broad spectrum
antibiotics.
Diphtheria bacilli arc
anrigeideally heterogeneous. By agglutination,
gravis strains have been classified into 13 types,
intermedium into 4 types and mifis into 40 types.
Gravis strains of types I and III have been reported
to be comment in Great Britain, type II worldwide,
type IV mainlv in Egypt .and type V in the USA.
No connection has hecn established between type
specificity and other characters.
About 15
bacteriophage types have been described. Type T
and III strains arc mitis. IV and VI intermedins,
VII avirulent gravis and the remainder virulent
gravid The phage types are apparently stable. A
system of bacterincin (diphtheriem) typing has
also been described. Other methods of typing
include bacterial polypeptide analysis, DNA
restriction patterns and hybridisation with DNA
probes.
The incubation period in
diphtheria is commonly 3~4 days hul may on
occasion be as shore as one day. In carriers, the
incubation period may be very prolonged. The site
ol infection may he (1) faucial; Q) laryngeal;
(3) nasal; {4) otitic; (5} conjunctival; (6) genital^
vulval, vaginal or prcpudal; sod (7) cutaneous,
which is usually a secondary infect ion on pre­
existing skin lesions. Sometimes diphtheritic
whitlow or ulcer may occur. Cutaneous infections
are commonly caused by nontoxigenic strains of
diphtheria bacilli.
Faucial diphtheria is the commonest type and
may van' from mild catarrhal inflammation to
verv widespread involvement. According to the
clinical severity, diphtheria may be classified as:
1. Malignant or hyperto\k i n which there is severe
toxemia with marked adenitis (bullncck). Death
is due to circulatory failure. There is high
incidence of paralytic sequelae in those who
ret over.
2. SeplLC, which leads to ulceration, cellulitis and
even gangrene amurd the pseudomembrane;
and
3. Hem orrhagic, which is characterised by
bleeding from [lie edge of the membrane,
cpistaxis, conjunctival hemorrhage, purpura and
generalised bleeding tendency..
The cumEnun complications are:
L Asphyxia due to mechanical obstruction of the
respiratory passage by the pseudomembrane for
C o p y rig h te d m aterial
 * Corynetocterium ►
which an emergency tracheostomy may become
necessary.
2■ Acute circulatory failure, wh:.ch may be
peripheral or cardiac,
3. Ptastdiph iheri ric paralysis, whic h typically occurs
in the third or fourth week of the disease;
paJatinc and ciliaiy but not pupillary paralysis
is characterise , and spontaneous recovery'is the
rule.
4. Septic, such as pneumonia and or-ris media.
Relapse may occur in abour 1% of cases,
Diphtheria is a toxemia. The bacilli remain
routined to the site of entry, where they multiply
and form the to\in.Thc to\i:i causes local nccrofc
changei and the resulling fibrinous exudate,
together with the (Iintegral Lug epirheli.Ll cells,
leucocytes, CTythrocytes a nd bactcri ,i, const irutc the
pseudomembrane, which is characteristic of
diphtheritic infection. The mechanical
complications of diphtheria are due to the
membrane, wh:k the systemic effects arc due to
the toxin.
Nonlmigenic Ktr.iins of diphtheria hac iILi may
cause infection even in immunised individuals, as
immunity with the toxoid does not confer
antibacterial immunity. Such infection is mild
tb<mgh pseudomembrane formation may sometimen
occur.
Diphtheria does not occur uaturailv in animals
but infection can be produced experimentally.
Susceptibility varies in different species,
Subcutaneous inoculation of a guinea pig with a
culture of virulent diphtheria bacillus will cause
death in 1—4 days. At autopsy, the followingfeatures
can be observed:
1. gelatinous, hemorrhage edema and, often,
necrosi s at the site of inoculation;
2. !uvollen and congested draining lymph nudes;
3. peritoneal exudate which may be dear, cloudy
or bloodstained;
4. congested abdominal viscera;
5- enlarged hemorrhagic adrenals, which is the
pathognomonic feature;
235
6- clear, cloudy or bloodstained pleural exudate;
and
7. (HHiietlines, p ericard ial effu sio n .
L a b o ra to ry d ia g n o s is Laboratory
confirmation of diphtheria is necessary for the
initiation of control measures and for
epidemiological purposes but nut fur the treatment
of individual cases. Specific treatment should be
instituted j'jnjTiftirareJy on suspicion of diphtheria
without waiting for laboratory tests. Any delay may
be fatal.
Laboratory diagnosis consists of isolation of the
diphtheria bacillus and demonstration of its toxicity.
One or two swabs from the lesions are collected
under vision, using a tongue depressor. Diphtheria
bacilli may not always he demonstrable in smears
from the lesion, nor car thev be confidcntiv
d"iiferen ti ated from some commensal cotyncbactcria
normally found in the rhroat. Hence smear
examination alone is not sufficient for diagnosing
diphtheria but is important in identify ing Vincent's
angina. i'br this, a Gram or Ln-hmatl stained smear
is examined lor Vincent’s Rjiinochetes and fusiform
bacilli. Toxigenic diphtheria bacilli may be identified
in smears by immunofluorescence. For culture, the
swabs are inoculated on Lexifier's serum slope, tellurite
blood agar and a plate of ordinary blood agar, the last
for differentiating streptococcal or staphylococcal
pharyngitis, which may Simulatie diphtheria. If the
swab cannot be inoculated promptly it should be kept
moistened wir h sterile serum so that the bacilli remain
viable. The serum slope may show growth ml 4-B
hours but if negative, will have to be incubated for 24
hours. Smears stained with methylene blue or one of
the speci.il stains (Ncisscr or Albert stun) will show
the bacilli with metachroitia:ic granules and typical
arrangement.Tellur lie plates will have to be incubated
fer i ‘, least two days lxl.ire :ieiuj[ considered nejptive,
as growth may sometimes be delayed. The tellurite
medium is particularly important in the isolation of
diphtheria bacilli from convalescents, contacts and
carriers as in these cases they mav he outnumbered
by other bacteria.
C o p y rig h te d m aterial

Any Isolate of the diphtheria bacillus should he
tested fut virulence or lOKigcnieity for the
bacteriological diagnosis re> be complete. Virulence
testing mar be bv in vivo or in vitro methods, the
former by the subcutaneous or intradermal test and
the ]jitter by [he precipitation rest or the [issue
culture test.
1' : *■ :
The growth from an
overnight culture on ^neffler'1- slope i=
emulsified in 2 -4 ml broth and O.R ml of the
emulsion injected subcutaneously into two
guinea pigs* one of which has been protected
with 500 units of the diphtheria antitoxin
13-24 hours previously. If the strain it virulent,
the unprotected ammal will die within four days*
showing the autopsy appearance described
earher. The method is nor usually employed as
it is wasteful of animals
The broth emulsion of
the culture is inoculated imracutaneously into
iwu guinea pigs (or rabbits) so [hai each receives
0.1 [ T i l in two different hiten. One animal acts us
the control and should receive1 500 units of
antitoxin the previous day, iTe other is given
50 units of antitoxin mtraperitoneally four hours
atrer the skin test,. in order to prevent death.
Toxigcnitiry is indicated by inflammatory
reaction at the site of injection, progressing to
necrosis in 4S-72 hours in the test animal and
no change in the control animal. An advantage
in the inrracutaneous test is that the animals do
not die. As many as ten mains can he tested at a
time on a nhbir.
dish while the medium is still fluid. If horse
scrum is not available, sheep ot rabbit scrum
may he used. When the agar ha* net, the surface
is dried and narrow streaks of rhe strains are
made at right angles to the filter paper strip. A
positive and negative control should be pot up.
The plate is Locuhated at 37 for 24-48 hours.
Toxins produced by the bscterf.il growth will
diffuse in the agar and where it meets the
antitoxin at optimum concentration will produce
a line of precipitation (Fig. 26,2)- The presence
of such arrowhead lines of precipitates indicates
that Lite strain is toxigenic. No precipitate will
furtil in the case uf nonloxigenic strains. This
test is very convenient and economical hut some
brands of peptone and some samples of scrum
do not give satisfactory results.
The to x ig e n id ty n f
diphtheria bacilli can be demonstrated by
Lncorpoiating the strains in the agar Overlav of
cell culture monolayers. The toxin produced
diffuses into the cells below and kills them.
Diphtheria was formerly an

,t ‘ fi'.T: :.iru
: A rectangular
strip oflilter paper impregnated with diphtheria
antitoxin ( 1GOOunit^m I) is placed on [lie surface
of a 20% normal horse serum agar in a petii

C o p y rig h te d m aterial
 i C o ry n -1J aci£riuni ►
important pediatric disease all over the world but
followi ug the development of effect ive prophylat rics
and mass Immunisation, the disease has been
virtually eradicated from most advanced countries.
In those developing countries where childhood
immunisation programmes have been implemented
effectively, diphlherl.i has become rare but in others
it continues to be a serious problem. The prolonged
and extensive epidemic of diphtheria in parts of
the erstwhile Soviet Union in the 1990s, involving
several thousands, with a mortality of up to 20 per
Lent is a warning of what can befall countries that
neglect immunisation and let living conditions
deter iorate-
ln endemic areas., it is mainly a disease of
childhood. It is rare in the first year of life due to
the passive immunity'obtained from the mother,
reaches a peak between two and five years, falls
slowly between five and 10 years, and rapidly
between 10 and 15 years with only verv low
incidence afterwards because of active immumtv tf
acquired hy repeated subcliniical infections.
Asymptomatic carriers who outnumber cases by
a hundredfold or more in endemic areas are the
most important source of infection. In the temperate
rc.'iwn-. carriage i- mainly in the nose and throat.
Nasal carrier harbour the bacilli (or longer periods
than pharyngeal earners. In the tropics, diphtheria
baciHi \nfeet the --kin more often tlian the respiratory
tract-Tosigcnic diphtheria bacilli may persist in
the skin (or over thTee years, Cutaneous infections
■nay stimulate natural immunity to diphtheria but
may also lead to fatici^l diphtheria In non immune
contacts, Fomites do not seem to play an important
role though in special situation toys and pencils
mav act as vehicles ol infection.
In nature, diphtheria is virtually confined to
human beings, though cows may on occat-iun be
found to have diphtheritic infection of the udder-
The infection in such cases is invariably transmitted
bv the milker. The L:ift*.r i■ maybe spread through
tlie milk of infected cows.
23?
pROPHTt l v m ?;
Diphtheria can be controlled by immunisation.
Three methods of immunisation are av:ulab-let
active, passive and combined. Of these, only active
immunisation can provide herd immunity and lead
to cradicaf ■m of the disease. Pas-.: vc and combi ned
immunisation can only provide emergency
protection to susceptible individuals exposed to ri -l-c_
The objective of : immunisation Is to increase
protective levels of antitoxin in circulation. Early
in the development of diphtheria prophylactics*
when immunising agents were scarce and not free
from ride, it was customary to test for susceptibility
before active immunisation was given. The
susceptibility test used was the Schick test
■ntroduced in 1913, The Schick test is no longer in
use, (Older editions of this textbook may be
consulted for derails of Schick test, if required).
The availability of safe and effective toxoid
preparations has made susceptibility tests
unnecessary. If, for any special reason, the
circulating antitoxin level is to be assayed, it can
now be done by serological test? such as passive
hemagglutination or by neutralisation in cell
culture. Antitwir level of 0-01 unit or more per ml
of blood is considered as index of immunity.
A c tiv e im n m n ism in n : Diphtheria
immunisation In children was initiated in 1913 by
von Behring ui-lng a toxin-antitoxin mixture
containing only enough anatoxin to leave the toxin
just underneutrailsed. This was a hazardous
preparation because the final tonicity of such
mixtures was determined not merely by the relative
proportions rtf toxin and antitoxin but also by other
variables, including the maimer ici which the toxin
and antitoxin were mixed. If to a given amount nf
antitoxin, the equivalent amount of toxin was added
all at once, the mixture remaned nontoxic. If instead,
the same amount of toxin was added in two or more
instalments at intervals of 1? minutes or mo-re, the
resultant mixture was toxic. This paradoxical
occurrence known a-1- the Danyyz nhcnamenm
C o p y rig h te d m aterial
and was due to the ability ot the toxins and antitoxins
tci combine in vatying proportion?, When the toxin
was added in instil clients, the toxin added 11: - r
combined with more than it-: equivalent of antitoxin,
laving insufficient antitoxin behind to neutralise
the toxin added subsequently.
Several other preparations were introduced for
active immunisation (for details, the third edition
of tills textbook may be consulted). Only two
preparations arc in llhc now, Form al Eusoid (also
known as fluid toxoid) and adsorbed toxoid- For mol
toxoid is prepared by incubating the main with
formalin at pH 7.4 7.6 for three to four weeks at
37 "C until the produce is devoid of Toxicity while
retaining i mmu nogeoici tv. Adsorbed toxoid is
purified toxoid adsorbed onto insoluble aluminium
compounds— uyu.illc aluminium phosphate, lefis
often the hydroxide. Adsorbed toxoid is much more
immunogenic than the fluid toxoid. Due to anxiety
about an enhanced risk of provocative poliomyelitis,
it wan replaced by fluid to x in d for Mime tim e.
I lowcvtr, as the risk is considered small and as the
Lrnmunogcnidty of fluid toxoid was unacceptably
low, adsorbed toxoid is now used almost universally
as the preferred agent. It is advisable to give
adsorbed toxoid hy intramuscular injections as
subcutaneous injection may be painful,
Diphtheria toxoid is usually given in children
as a trivalcnt preparation containing tetanus raxoid
and pertussis vaccine also, as the DTP, D PI' or
triple vaccine. A quadruple vaccine containing in
addition the inactivated poliovaccinc is also
available. For young children, diphtheria toxoid
given in a dose of 10-25 Lf units is recommended.
Smaller doses (1 -2 L f units) are used for older
children arid adults to minimise adverse reactions.
In toxoid preparations the lower dose of toxoid is
indicated by the small letter’d' and the full dose by
capital LDf. For example, the tetanus diphtheria
vaccine for adults containing low dose diphtheria
tOXuid is referred to as 'Td'.
The soltedule of primary immunisation of infants
and children consists of three doses of DPT given
at intervals of at least toor weeks, and preferable
six weeks or mom, followed by a fourth dose about
a year afterwards. A further booster dose is given
at school entrv.
This is an
emergency measure to be employed when
susceptible? are exposed to infection, as when a case
o f diphtheria is admitted to general pediatric wards.
It consists of the subcutaneous administration of
500-lQQti uni ts. of antitaxi n font idiphtberi tit serum,
A1 IS). A? this is a horse scrum, precaution against
hypersensitivity should be observed.
This consists of
administration of the first dose of adsorbed toxoid
on one arm, while ADS is given on fhc other arm,
to be continued by the full course of active
immunisation. Ideillv, all canes that receive ADS
prophylactically should receive combined
immunisation.
The toxin cu-ntent of culture filtrate? varies
cohmJ iirablv from batch Co batch. As such their
standardisation or measurement should be with
reference to rheir biological activity. Ehrlich
defined the minimum lethal dose (M LD) of the
diphtheria toxin as the least amount of the toxin
required to kill a guinea pig weighing 250 g within
% hour? after subcutaneous inoculation. One unit
of antitoxin was defined as the smallest amount of
antitoxin required to neutralise 100 MLD of toxin.
Keeping a labile substance like the toxin as the
standard lei] To inaccuracies. Toxin undergoes
spontaneous, deraturatior into toxoid which will
combine equally well with the antitoxin, Thus, any
sample of toxin trill contain a variable quantity' of
toxoid which will vitiate standardisation of
antitoxin. The antitoxin, on the other hand, is
permanently Stable in the treeae-*-dried state.
Therefore, the antitoxin has heen adopted as the
reference preparation. Ehrlich? original antitoxin
is accepted as the international standard. One
C o p y rig h te d m aterial
antitoxin unit (AU) is defined as die amount of
antitoxin that has the $ante tout combining capacity
far toxin and toxoid together as one unit of Ehrlich's
original iruitoxin.
Since toxin always contains some toxoid, two
other unifH for measurement of toxin have Keen
introduced, the LG and L+ doses. The LO (Limes
nul) dose of the diphtheria toxin is the largest
amount of toxin that, when mixed with one unit of
antitoxin and injected subcutaneously into a 250 g
guinea pig, will on the average cause no observable
reaction. As 'no reaction’ is nor a definite end point,
in actual practice, the end point is taken as minimum
local edema. TTic L* (Limes ttxJ) dose ol diphtheria
toxin is the smallest amount of toxin that when
mixed with one unit of antitoxin and injected
subcutaneously into a 250 g guinea pig will on the
average kill the animal within 96 hours. If toxin
combines with antitoxin in constant proportions, it
would be expected that the difference between the
Ia dose and the I Si dose would be equal to 1 MI ,D.
But when the estimations are actually made, it is
found to vary from 10-100 MLD or more. This
discrepancy is due to the presence in toxin
preparations of varying amounts of toxoid and to
the ability of rhe toxin and anriroxin to combine in
varying proportions. This is known as the Ehrlich
phenomenon.
The use of death as an end point for the titration
of toxin is wasteful of animals. Romer introduced a
method of titration employing the erythematous
swelling produced by the intndetmil injection of
toxin, and its neutralisation by antitoxin. The
minimum reacting dose (MRD) is the Ica^ramounr
of toxin that when iniected intradermally in a guinea
pig, causes an erythematous- Hush 5 oim in diameter
visible alter 36 hours, rrhe I dose is the smallest
t the
amount of toxin which, after mixing with 1 unit of
antitoxin, will produce a minimal skin inaction whe n
injected irUrudern Lilly into a guinea pig.
Ramon introduced a test tube method fo r
titrating toxin and antitoxin based on flocculation.
The flocculating or Lf unit of diphtheria toxin is
the amount of toxin which flocculates most rapidly
with one unit of anriroxin. The Lf unit has several
advantages. It is inexpensive and rapid and docs
not need immak. it is also the only method available
for the titration of toxoids. The amount ol toxoid
in prophylactics is expressed in L f unit^.. Many
other in vitro teats have been developed for antigen
assay. These include cell culture neutralisation tens
using rabbit kidney cells, jussive hemagglutination
test with roxi n coated ta nned sheep RKC, and HI A-
Sptcifjc treatment of diphtheria
consists of anti toxic and antibiotic therapy, Antitoxin
should be given immediately when a case is
suspected as diphtheria, as the fatality rate increases
with delay in starting antitoxic treatment. The
dosage recommended is 20,000 to ] ,00,000 units
fire serious ease:;, half the dose being given
intravenously. Antitoxin treatment is generally not
indicated in cutaneous diphtheria as rhe causative
strains are usually nontoorigmit-
C- diphthcrine is sensitive to penicillin and can
be cleared from the throat within a few days by
penicillin treatment. Diphtheria patients arc given
a course of penicillin though it only supplements
and docs not replace antitoxin therapy,
Etythromycin Is more active than penicillin in rhe
treatment of carriers.
This bacillus related to G, d ip h th eria c can cause
diphtheria-like Lesions. It resembles the gravis npe
of the diphtheria bacillus hut it liquefies gelatin,
ferments trehalose slowly and docs not reduce nitrate
to nitrite. It produces two types of toxins, one
prohably identical with the diphtheria toxin and
other resembling the toxin of
C. pscudotubcFculosis. It is pathogenic to guinea
pigs, the lesions produced resembling those caused
bv C , d ip h th e r iitc . It lias been found to cause
infection in cows, and human infections may be
transmitted through cow's milk, II is sensitive To
erythromycin, Diphtheria ami toxin is protective.
C o p y rig h te d m aterial
It has been suggested That C, uibersns may be
considered a subgroup of diphtheria liaeillL rather
than a separate species.
Arcanobactenum (formerly Corynebactcrium)
hacinol\ticunn:j.n. cause pharyngitis and. skirt ulcers.
C.jakduni can cause cutaneous and blood infeetin tns
m immunocompromised hosts. I t is usually
multi resistant, responding only to vancemydn.
Cofynebacierb of veterinary importance are rbc
PrcisT Nocard bacillus (C. pycuilotubcrcuSiNSis);,
which causes pseudo Tuberculosis in sheep and
suppurative Lymphadenitis in horses, C. ntnaJc
causing cystitis and pyelonephritis in cattle and
C, equf, isolated from pneumonia in foals.
and other areas. These may sometimes be mistaken
fur diphtheria bacilli and are called diphtheroids.
Ln general, diphtheroids stain more uniformly than
diphtheria bacilli, possess few or no mccachromatic
granules and [end Eu be arranged in parallel rows
(palisades) rather than cuneiform pattern. However,
some diphtheroids may be indistinguishable from
diphtheria bacilli microscopically Differentiation
is by biochemical reactions and more reliably by
virulenec tests. The contnion diphtheroids are
C. p.'xudodiphtheritiL-uin (C. hafnwnnii) found in
the throat and C. jremsj.s found ir the conjunctival
sac. The former is urease positive and does not
ferment glucose, while the latter is uncase negative
and terments glucose. Both are pjTar.inamLdasc
positive, unlike diphtheria bacilli.

Erythrasma, a Localised infection of the stratum O r * : k . CeftYWiEPSHMC

eomeum usually affecting the axilla and groin, is Besides genus G>rtTiehacrertujHNa. number £>fOther
caused by C mj'nufrs.dmjjm. This is a lipophilic genera of coryncform bacteria have been defined.
corynebaeterium and can be grown readily in media Among them, the genus Piopiooibdctenutn is of
containing 20 per cent fetal calf scrum. medical interest as three species, fl aeries, R
C. tenuis has been associated with trichomycosisgn n u lo su m and R tvidum , are consrantiy present
axillaris, characterised by the formation of on human skin.Thev are anaerobic and ,ierotolerant;
pigmented nodules around axitlary and pubic hair growing well in lipid containing media. P- arjie.s is
shafts. often isolated from acne lesions bur its pathogenic
rolr is uncertain.
Corfc-nebjL-rej-JUiTT purvum which is frequently
Coiyncbactcria resembling C. diphtheria? occur a.s used as an immunomodulator is a mixture of
normal commensals in the throat, akifl, conjunctiva PropLombacterium species.

Coyle MB and BA Lipjly 1990. CorrneMtm bacteria in infectious disease. Clift Microbiol Rev 3:227.
Elardy ERBtt j L. IWh. Resurgence of diphtheria in the farmer Soviet Union. Lancer 347:1991.
Hoflrr W, 199]. Cutaneous diphtheria, h t t j Ltmufcd, 3Q:ftJ5.
j£ami n 1.1 Corynebjctrr u^i. 1n Colics, JG et i’ fed';). Practical M nlical M jc ra fw ijg r. 1J" edn. Edinburgh: Churchill-
Lirii^mK.

C o p y rig h te d m aterial
 Bacillus
Sporogenous, rod-shaped bacteria arc classified
into two gcner.i, tEit: aerobic HaciHr and the
UlflEiubic C loutriiiix. The genus B acillus cons isitu
of aerobic bacilli forming heat resistant spores. I"bcy
arc Gram positive but tend to be decolourised easily
so as to appear G ram variable, or eve n frankly G ram
negative. They are generally mot lie with
peritrichous flagella, the anthrax bacillus being a
notable exception, Members of this group exhibit
great diversity in their properties. The genus
includes psych rophilic, mcsophilic and
thermophilic species, the maximum temperatures
for vegetative growth ranging from about 25 ' C to
above 75 °C and the minimum from about 5 CC to
45 "C. Their salt tolerance varies from less than
2% to 25% NiCl.
Their sports are ubiquitous, being found in soil,
dust, water and air and constitute the commonest
contaminants in bacteriological culture media.
fkiL-jVjLrs jrnthfijCrs, the causative agent of anthrax,
is the major pathogenic species. Ji. corns can cause
ftMuibome gastroenteritis. Some species may lie
responsible for opportunistic infections.
Considerable historical interest is attached to the
anthrax hacilluR. It was the first pathogenic
bacterium to be observed under the microscope
(PbUendcf, 1849), the first communicable disease
shown to be transmitted by inoculation of infected
blood (Davainc, I 8S0), the first bacillus Co be
isolated in pure culture and shown to pissess spores
{Koch, 1876) and rhe first bacterium used for the
preparation of an attenuated vucc] ilc(Platen r, 1881).
Anthrax was for long feared as a potential tool
in biological warfare. This fear became an actual
fact in 2001, when anthrax in the form of Weapons
grade spores, having enhanced dispensability and
virulence was sent hy mail to various destinations
in the USA, causing disease and death in many
persons.
The anthrax bacillus is one of the
largest of pathogenic bacteria, measuring 5 -1 0 flm
* 1-1 -fi |Jlm. In tissues, it is found singly, in pairs or
in short chains, the entire chain being surrounded
by a capsule. The capsule is polypeptide in nature,
being composed of a polymer of d(-) glutamic acid.
Capsules arc nut formed under ordinary conditions
of culture hut Only if the media contain added
bicarbonate or are incubated under 10-25% CO,.
If grown in media containing serum, albumin,
charcoal or starch, capsule formation may occur in
the absence of CO,.
Irt cultures. the bacilli are arranged end to end
in long chains.The ends of the bacilli arc truncated
or often concave and somewhat swollen so that a
chain of bacilli presents a "bamboo Stick’ appearance.
Spores arc formed in culture or in the soil but never
in the animal body during life Sporulation occurs
under unfavourable condition* for growth and is
encouraged by distilled water, 2% NaCl or growth
in oxalated agar. Oxygen is required for spondatiem,
but not for germination, Spoliation is inhibited
by calcium chloride. Spores are central or
subtermin.il. elliptical or oval it] *hape, and ,Lrc of
the same width as the bacillary body so that thev
do not cause bulging o f the vegetative cell (Fig.
27.1).
C o p y rig h te d m aterial
 while avitulcnt or attenuated omi t form smooth
colonics. Oil gelatin stall . Iruri a characteristic
‘inverted fir tree' appearance ■ seen, with slow
liquefaction commencing from - top (Fig. 27.3),
O n hlood agar, the cold rues !■ nOnlieniolytic1
rhough occasional strains ■■ ■■ a narrow Tone
o f hemolysis. In broth gtuWtlL • as fiotCLdu
deposit, with little or no t rbidjty.
When B. anthnds is grown i the surface of a
solid medium containing il5 ■' ' ) units of
pcnLciillJ ri/ml, in 3—6 hours the cell become large*
spherical, and occur i tains on d surtace of the
agar, resembling a string of pc iris. This 'string of
■ r pearta reaction different late:- . ■ i:! B. Aitthttds
From B. ccfcus and other acrobh spore bearers.
The anthrax bacillus is Gram positive and
Another useful test to . ItttiaTr between the two
nonacid fast, The spores do noi stain by ordinary
is the susceptibility ot'£#, ■ gamma phage,
methods hue can be stained diffrrendally by special
A selective rt]edLum (P L E T r c Imi: ■-. consisting
techniques. When stained with Sudan black b hEat
of polymyxin, lysozyme, diamine tetra
globules may be made c]ut within the bacilli. When
acetic acid (EDYA) and r acetate added to
blood films containing anthrax bacilli arc stained
heart infusion agar, hai been ■ to isolate B.
wirh polychrome methylene blue for a tew seconds
anthneis from mixtures emta n nu other spore-
and examined under the miauttOpCj an amorphous
bearing bacilli.
purplish material is noticed around The bacilli.This
represents the capsular material and is characteristic
o f the anthrax bacillus. This is called the
M’Fndy can’s reaction and is employed for the
presumptive diagnosis of anthrax in animals*
'Hie anthrax bacillus is nonmntile* unlike most
other members of this genus,
■ ■ It is an aerobe*
.Mid Ji facultative anaerobe, with 1 temperature tjaijte
for growth of 12-45 °C (optimum 35~37 aO - The
optimum temperature for sporulation is 25-30 7C,
Good growth occurs ou ordinary media.
On agar plates, irregularly round colonies are
formed, 2-3 mm in diameter* raised, dull, opaque*
greyish white, with a frosted glass appearance,
Under the low power microscope, the edge oi the
colony is compiled of Jong, interlacing chains of
bacilli, resembling locks of matted hair. This is
Called the 'Medusa head appearance’ (Fig. 27.2), i
V irulent eapsulitcd strains torm rough cultures*

C o p y rig h te d m aterial
 Glucose, maltose and
sucrose arc fermented prodding acid but no gas.
Nitrites are reduced to nitrites Catalase is formed,
■ The vegetative bacilli are riot
particularly resistant and are destroyed at hi) QC in
30 minutes. In the carcasses of animals which have
died of anthrax, the hsciJli remain vjah]e in the
bone marrow for a week and in the skin for two
weeks. Normal heat fetation of smears may nut kill
the bacilli in blood filmSr The spores arc highly
resistant to physical and chemical agents. They
have been isolated from narurally infected soil after
as long as 60 years. They resist dry heat at 140 *C
for 1-3 hours and hoiling for If) ml notes. I'hcy
survive in 5% phenol for weeks. H g C l; in a 1/
1000 solution may tail to kill anthrax spores in less
than 70 hours. Four per cent potassium
permanganate kills them ia 15 minutes, Destruction
of the spores in animal products imported into
nonendcmic countries is achieved by 'duckering' in
which formaldehyde is used as 2% solution ar
3(K40 QC for 20 minutes for disinfection of wool
and as 0 .2 5 per cent at 60 gC for nix hours for lesion, heart blood and spleen (more than 10* bacilli/
animal hair and bristles. The anthrax bacillus is ini). The bacilli am teen confuted to ihe interior
susceptible to Sulphortumi Jes, penicillin,, of the capillaries, where their numbers may be so
erythromycin, streptomycin tetracycline and great as to obstruct the flow of blood.
chloramphenicol. Occasional strains resistant ro Two virulence factors have been identified—
penicillin haw been met with. the CAf1}■uj'jitpflh7Kptsdt? and the Ail dim* Sarin, each
In nature, anthrax is primarily o t which is encoded by a separate plasmid.
a diseaie of cattle and sheep, and less often of horses The capsular polypeptide aids virulence by
and swine bur experimentally most animals are inhibiting phagocytosis. Loss of the plasmid {p*02J
susceptible to a greater or lesser degree. Rabbits, which control* capsule production Leads to lots of
guinea pigs and mice are susceptible. Infection can virulence. This is how rhe live attenuated anthrax
be produced with difficulty in birds. Frogs are spore vaccine (bteme strain) was obtained,
resislant, while toads are wry susceptible. Anrhmx toxin was identified by the finding that
Following the subcutaneous inoculation of a die injection of sterile plasma of guinea pigs living
culture into a guinea pig, the animal dies in 24-72 of anthrax into healthy guinea pigs killed them and
hours, showing a local, gelatinous, hemorrhagic that death could be prevented by immune scrum.
edeot.L at the site of inoculation, extensive The toxin is a complex of throe fractions: die edema
subcutaneous congestion and characteristically, an factor (OF OT Factor I)hthe protective antigen factor
enlarged, dark red, friable spleen. The hlood is dark (PA or Factor I [) and the lethal factor (L F nr Factor
red and coagulates less firmly than normally. The IJ1), They are not toxic individually but the whole
bacilli are found in Large numbers in the local complex, produces local edema and generalised

C o p y rig h te d m aterial
344 i Tc*tbcok d Microbiology
shock. The three factors have been characterised
and cloned- PA IS the fraction which hinds to the
receptors on the target cell lurfoce, and in turn
provides airadimcnc sites for O F or I,F, facilitating
their entry into the cell. Antibody to FA is protective
because it blocks the first Step 1:1 [OXin activity,
namely its binding to target cells. Oh is an adenyi
cyclase which is activated only inside the target
cells, leading to intracellular accumulation of cyclic
AMP. Tliis Is believed to be responsible for the
edema and other biological effects of the toxin.
Entry of LF into the target cell causes cell death
bur the mechanism of action is not known. Loss of
the plasmid (p>dll) which encoder the anthrax Cunnr
renders the strain aviruIcnt.This is believed m have
been the basis for the oil gin al anthrax vaccine
developed by Pasteur. The aviruLent Sterne vaccine
Strain in devoid of the plasmid coding for [Lie
capsular polysaccharide.
A nthrax
Anthrax is a 7jnunnsis. Animals are infected by
ingestion of the spores prcscru in the soil. Direct
spread front animal to animal Is rare. iTc ih-casc
is generally a fatal sept iCemi.1 but may some limes
be localisedh resembling the cutaneous disease in
human beings. Infected animals shed in the
discharges from the mouth, nose and rectum, large
numbers of bacilli, which sporulatc in soil and
remain as the source of infection.
Human anthrax is contracted from animals,,
directly or indirectly- The disease may be (l)
cutaneous; (2) pulmonary; or (3) intestinal, all types
leading to fatal scptii emu or meningitis.
C utaneous an th rax follows entry of the
infection through the skin-The lace, neck, hands,,
arms and back arc the usual sires. The lesion starts
as a papule 1-3 days after infection and becomes
vesicular, containing fluid which may he dear or
bloodstained. The whole area is congested and
edematous, and several satellite lesions filled with
serum or yellow fluid are arranged round a central
fieerol:’-1 lesion which is covered by a Hack eschar.
*■
{The name anthrax, which means coal, comes from
the black colour of the eseliar.) The lesion is called
a ma Jigj\nntpushik ■.The 1 1iscase used to be common
in dock worker* carrying loads of hides and skins
on their bare backs and hence was known as the
firde /Kvrcrs disease. <Sutareons anthrax generally
resolves spontaneously, but 1 0 -2 0 per cent of
untreated parients may develop fatal septicemia Or
meningitis.
Pulmonary anthrax is called the i™ J sorters
disease because if used to be common in workers
in wool factories, due to inhalation of dust from
infected wool. This is a hemorrhagic pneumonia
with a high fataluy rate. Hemorrhagic meningitis
may occur as a complicarinn.
Intestinal anthrax is rare and occurs mainly in
primitive communities who eat the carcasses of
animals dying of anthrax, A violent enteritis with
bloody di.inhca occurs, with high case fatality.
Human anthrax may be industrial or non­
industrial (agricultural). The former is found in
workers in indusliies such as meat packing or wool
factories. Mon-industri*1 anthrax is often an
occupational disease in those who associate
frequently wirh animals, such as veterinarians,
butchers and formers. Tt may also he found in the
general population. Cutaneous anthrax used to he
caused by shavLng brushes made with animal hair.
Stomoxvs calcitnms and other biting insects may
occasionally transmit infection mechanically.
Anthrax IS enzootic in India, the number o f
ar.imah infected running into lens o f thousand*
annually. The disease is rare In some countries such
as Britain, where infection is imported through
contaminated hides, hone meal fertiliser and other
animal products-The extent of anthrax in human
beings is nor dear but about 20,000 m 100,000
cases are believed to occur annually throughout the
world, mostly in rural areas. Large epidemics of
anthrax were reported from Russia and Zimbabwe
■.Ili r.i il; 1978—SO. An epizootic o f anthrax ml sheep
has been active near the Andhra-Tamil Nadu
border^ causing many cutaneous and
C o p y rig h te d m aterial
 4 H a d Hus ►
nteningoencephaiiuc human infections, with high
mortality rate. There have heen outbreaks in
Karnataka and West Bengal- Anthrax infection in
human beings provides permanent immunity and
second attacks are extremely rare,
[ . 53h<irnl(trv diilgnoftiti A nthrax may be
diagnosed by m i .:ru atu py, culture, animal
inoculation and serological demonstration of the
anthrax ami gen in infected rissues. Acute and
convalescent phase sera should be obtained, since
anti bud lCs Co the oiganKmCan be demoruitrated by
gel diffusion complement fixation, arefigen coated
tanned red Cell agglutination and E L IS A
techniques, The type of test to be employed depends
on the nature of the material available.
When an animal is suspected to have died of
anthrax, autopsy is not permissible, as the spilt blood
uiil lead Co contamination of the soil. An ear may
be cut oft'from the carcass and sent to the Laboratory.
Alternatively, swabs soaked in blood or several
blood smears may be sent, The demonstration of
G rim positive bacilli with the morphology o f
anthrax bacilli and a positive M'Fadyearfs react ion
will enahle the presumptive diagnosis to he madr.
Immunnfluorescent microscopy can confirm the
identification. Isolatium of the hac iILus i-j easy it gross
contamination has not occurred. The anthrax
bacillus can often be isolated from contaminated
tissues by applying them over the shaven skin erf a
guinea pig. It is able to penetrate through minute
abrasions and produce fatal: nfection. I f the sample
received is putrid so [hat viable bacilli art unlikely,
diignu--i- may be established bv A sco li’s
therm opretipitin test by demonstrat ion of the
anthrax antigen in tissue extracts.
Alter the hiotermrism experience in rhe USA
in 2001, the C D C (Centers for Disease Control)
have prepared guidelines In: identification of anthrax
bacillus. Any large Gram positive bacillus with the
general morphology and cultural features of
anthrax; nomnotile, nonhemolytic on blood agar
and catalase positive can he given a presumptive
report o f anthrax. For initial confirmation, lysis by
2 45
gammaphage and direct fluorescent antibody test
I D FA ) for capsule specific training and Mr
polysaccharide cell wall antigen are sufficient.
For a further confirm ifion, PC R lor anthrax
bacillus specific chromosomal markers can he done.
For epidem iological studies and strain
character isal ion, MLVA (nlull ipie locus variable
number tandem repeat analysis) or A FLP (amplified
fragment length polymorphism) can be used.
P r o p h y la x is Prevention o f human anthrax is
mainly by general methods such as improvement
of factory hygiene and proper ster ili-.u ion of ailimal
products like hides and wool. Carcasses of animals
suspected to have died of anthrax are buried deep
in quicklime or crem ated to prevent soil
contamination.
Prevention of anthrax in animals is aided by
active immulibation, rhe original Pasteur’s anthrax
vaccine is of great historical importance. It was
Pasteur s convincing demonstration of the protective
effect of his anthrax vaccine in a public experiment
at P ou illy -le-F u rt in lS S l that marked the
beginning o f scientific Lulniu no prophylaxis.
I'.islcijr'x vaccine was the anthrax bacillus attenuated
by growth at 42—45 nC-
As the spnne is the common infective form in
nature, vaccines consisting of spores of attenuated
strains were developed. The Sterne vaccine
contained spores of a noncapsulated avindent
mutant Strain- The Ma?Utchi vaccine cnnl.iined
spores of stable attenuated Carbazoo strain in 2%
saponm. The spore vaccines have been used
extensively in animals with good results. ] i-lx give
protection for a year following a single injection.
They are not con;-i deied sale for human use, though
they have been used far human immunisation in
Russia. Alum precipitated toxoid prepared from the
protec five antigen has been shown to be a safe and
effective vaccine for human use. It has been used in
persons occupationally exposed to anthrax infection.
Three doses intramuscularly at intervals of six
weeks between first and second, and six months
between second and third doses induce good
C o p y rig h te d m aterial
immunity which can T>Creinforced if necessary with
annual booster injections-
Antibiotic therapy i^ effective in
human cases but rarely succeeds in animals as
therapy Is not started sufficiently early. Antibiotics
haver no effect on the ton in once it is formed
Penicillin and streptomycin Hire no longer used for
treatment. They have been replaced by doJtyCU.-li[]e
and ciprofloxacin, winch are effective in prophylaxis
and treatment.
Many members of the genus bacillus, other [ban
the anthrax hacillus have occasionally caused human
infections. O f them, the most important is ft erneus
which from 1970 has been recognised as a frequent
cause of foodburne gastroenteritis. It has also been
associated with septicemia, meningitis, cndocandi tis,
pneumonia, wound infections and Other SUppuntiw
lesions, particularly as an opportunist pathogen. B.
subtilis, if. lichcnitorm is and a few nther species
have also been occasionally isolated from such
lesions. These and a large number and variety1 of
nonpathugem c aerobic spore bearing bacilli
appearing as common contaminants in cultures and
1, Nonmotik
2 Captukred
3, Grow in lopg chains
4. Medusa head colony
5. No growth in penicillin agar
(lfl umts/ml)
t i. Hemolysis absent or weak
7. Inverted tir tree growth and
slow gelatin liquefaction
8. N o turbidity jn broth
9. Salicin. fermentation negative
10. N o growth af 45 'C
] ] , Gm w ih Inhibited by chloral hydrate
12. Susceptible to gamma phage
13, Pathogenic to laboratory animals
having a general resemblance to anthrax bacilli have
been collectively called pitfildOitiilhrJx or lirt thraccuiJ
bacilli. 'Fable 27.1 lists the main differentiating
features between them,
ft census has become important as a cause of food
poisoning, lr is widely distributed in nature and
may be readily isolated from soil* vegetables and a
wide variety of foods including milk, cereals, spices,
meat and poultry, ft o n u s is generally monle but
nonmotile strains may occur. It resembles B.
m thttris except that it is not capsulated and nor
susceptible to gamma phage and docs not react with
anthrax fluorescent antibody conjugate. Animal
pathogenicity test also differentiates between the
two.
ft e-rim* produces two patterns of foodborne
disc ase. O ne is associated with a wide range of foods
including cooked meat and vegetables. It Is
ebaracreri-sed by diarrhea and abdominal pain,
H-lfi hours after ingestion o f contaminated foods.
Vomiting is rarc. ft ca su s is not found in large
numbers in feca! specimens from these patients.
The seCOnd type is associated almost exclusively
Generally motile
Noncapsulated
Grow in short chains
Not present
Grow usually
Usually well marked
Rapid liquefaction
Turbidity usual
Usually positive
Grows usually
Not inhibited
Not susceptible
Not pathogenic
C o p y rig h te d m aterial
 i Bacillus * 2 47

with the consumption of Looked rice, usually (Vied
rice from C ‘nncsc restaurants, Th^ illness ts
characterised by acute nausea and vomiring 1 -5
hours after [he meal. Diarrhea is not common,
fj. crretis is present i:i large number- in the cooked
ri.ee and fecal sample;; frnm these patients. Both
types of ill]ness are mild and self Limited,, requiring
no specific treatment.
It has been shown that the two types of disease
are caused by strains of B . cereus belonging to
different serotypes. The diarrheal disease is mostly
caused hy serotypes 2, 6, 8. 9, 10 or l i , while the
riicc associated emctii' illness Is caused by serotypes
] . 3 or 5. Isolates from the diarrheal type o f disease
produce an en terotoxin which causes fluid
accumulation in ligated rabbit ileaJloopHresembling
the hear labile enterotoxin of Escherichia < oii
Strains causing the emetic type of di-scasc produce
a toxin which causes vomiting when fed to Rhesus
monkeys, resembling staphylococcal enterotoxin,
Fhe emetic m dn was produced on Iv when B. Crreus
was gfuwn in rice hut not in other media. Two
mechanisms of action have been described for the
enterotoxin of B. ceneui, one involving stimulation
of C A M P system and the other independent o f it,
A special man mlled—egg yolk—phen □1 re d -
polymyxin agar (M Y PA ) mcdi'iim is useful in
i-olating B. ccrcus from fcccs and other sources B,
cereus produces lecithinase and ferments glucose
but not mannitol.

Further Rgftdiitg
Aksaray K et al. 1990. Cutaneous amhrax. Trcp Ceograph \ fed 42: 169.
brahman PS. 1980. Inhalation anthrax. A nn S ew York A cad Sci A53:B3.
Divon TC et a], 1999. Andirax. Sew E n g j Med 341:915
Indira Devi k. 2001. Anthrax J. Acad Clin Microb J5-:55.
L Jlih i MK and A Kumu 19%. Anthrax in South India. Lances 348:553.
LaforeeFM. 1994. Anthrax. Clin Jnirct Dtj 19:1009.
Mjt-d n. M et al 1994. The Sverdlovsk anthrax outbreak of 1979. Science 266:1202.
Mock M and A Foret 2u0l Andirax. A nn flevAfitrobjoJ 53:647.

C o p y rig h te d m aterial
 Clostridium
The genus Clostridium consists of Gram positive,
anaerobic", spore forming bacilli. The SpOfCS are
wider than the bacillary bodies, giving the bacillus
a swollen uppcare-nce, resembling a spindle. Hence
the name Clostridium (JtitwTer, meaning a spindle}.
The genus contains bacteria responsible tor three
major diseases of human beings - gas gangrene,
food poisr>ning and tetanus. Some o t the pat hogens,
tor example. Cl. pe-rfrmgpnM'aml CL re Mur are found
iiorin.iliv in human and animal intestine*. Many
species arc pathogenic hot most are saprophytes
found in soil, water and decomposing plant and
animal matter. Intestinal Clostridia rapidly invade
ilte blood ;iltd tissues of the host after death and
initiate decomposition of Lite cadaver. Some (for
example, CJ. ICCtabutylkvm) are nt industrial
importance, used for the production of chemicals
such as acetone and luitanol.
Clostridia are highly pleom orphic. They nre rod
shaped, usually 3-K pm * 0 .4 - 1 2, pm in size. Long
filaments and involution forms are common. Spore
formation occurs with varying frequency in different
sjiecies. Some [C l sporogents) sporulite readily
while others- (O . ywryrjrigrns] do tSO inconstantly.
Spondation takes place in the animal hody also.
The shape and position of spores vary in different
species and these are o f use in rhe identification
and classification of dostridia. 3 pure- may be:
1. Central or equatorial, giving the bacillus a
spindle shape [CL &&mttntenj}.
2. Subtenninl, the bacillus appearing club shaped
(Cl- pcriringzns).
3. Oval and terminal, resembling a tennis racket
[Q . tertJUTrt).
4. Spherical and terminal, giving a drums Lick
appearance [CL tefimj).
Closrridia ait motile with peritrichatc flagella,
with tew exceptions such a* CL pcrfiingcnv and
CL leLiui type VI which are nonmotile. The motility
is slow and h.L- been described as 'stately', C7,
pcrtringewa.ni] Cl. bu ttrfcum art capitulated, while
others are not.
Clostridia arc easily stained. They arc Gram
positive but in older cultures, cells are often Gram
variable, or even frankly Cram negative,
Clostridia are anaerobic. The sensitivity CO
o x y g e n varies in different species. Some (for
example, CL AOvyi) are exacting anaerobes and die
on exposure rn oxygen, while some others (for
example, CL histofyticum ) arc aeroiolemnt and may
even grow aerobic ally. More important than the
absence of oxygen is the provision of a sufficiently
low redox potential {l",h) in the medium, ITiis can
be achieved bv adding reducing substances such as
unsaturatvd fatty acids, ascorbic acid, glutathione,
cysteine, thioglycollic acid, alkaline glucose,
sulphites or metallic iron. A small concentration
of C O , appears to enhance growth. The optimum
temperature for pathogenic Clostridia is 37 'C .
Some saprophytic cLostridia are thermophilic and
others psyebmphylic. The optimum pH is 7-7.4.
Growth is relatively slow on solid media.
Colonial characteristics are variable. Some species
arc hemolytic on blood agar. A very useful medium
is Robertson's cooked meat broth. It contains
uiisnnj rated fatty acids which take up oxygen, the
reaction being catalysed bv hematiii in the meat,
and also sulphydril compounds which bring about
C o p y rig h te d m aterial
a reduced OR potential. Clostridia grow in the
medium, re rulering the broth turbid. Mott species
produce gas. SKcharolytic species rurn the near
pink. Proteolytic specie* turn the meat black and
produce toul and pervasive odours. In litmus milk
medium, the production of acid, clot and gas can
be detected.
T h u vegetative te lls o f dnstridiu do not differ
fro m ilo ns p o rin g b a c illi in th e ir re sista n ce to
physical and chem ical agents. The spores exhihir a
pronounced but variable resistance to Iteat, drying
and disinfectants, Spores o f Cl. botulinum survive
boiling after 5 - 4 hours and even at 105 C are not
killed Completely in less than 1DO minutes. Spores
o t m ost strains o f Q . perfhfngeji* are- destroyed by
boiling for less than five m inutes but spores of some
Type A strains that cause food poisoning survive
for scvernl hours. Cl- retnni spore* persist for yean
in dried earth. Spores o f som e strains of Gf. tetani
resist boiling for 15=^0 m inutes, though in most
Cases, they arc destroyed w ithin five m inute*. All
species are killed hy autoclaving aT 121 ‘C within
20 m in u tes. Spores arc particularly resistan t to
phenolic d isin fectan ts. Formaldehyde i* not very
active and spores may sometimes survive immersion
in 2% s-olutiori for upto five rial's. Hatngens are
effective and 1% aqueous iodine solution kills spores
w ithin three hours. Glutaraldehyde (2% at pH 7 .5 -
8.5) is very effective m killing spores. In general,
C lo strid ia a r e s u s c e p tib le to m e tr o n id a z o le ,
peiiLcill.ili, cep h alo sp o rin s and ch lo ram p h en icol;
lets so to tetracyclines, an d r e s is ta n t ro
aminoglycoside* and quinoLoncs.
C lostrid ia can produce disease or.lv when the
con d ition s are appropriate. T h e ir invasive powers
arc lim ited- Pathogenic clostridia form powerful
exotioxiiu. CL iwmi'jTUtn is virtually noninvasive and
iToninfectious. Botulism is due to the ingestion o f
preform ed toxin in food. CL teramhasLitkiavariwe
p roperty and is co n fin ed to th e p rim ary site of
lodgem ent. TetajLLLH results Irani the action o f the
p o te n t exotoxin it p rod u ce*. T h e g a* gangren e
Clostridia, besides being toxigenic, are ill so invasive
and can spread along tissues and even cause
septicemia.
Maiiy methods have been adopted for the
classification id' Clostridia. These include
morphological feature* such as the shape ami
position o f spores and biochemical features such
as saccharolytic and proreolyric capacities {Table
28,1), Clostridia of medical importance may abo
he considered under the disease* rhey produce {sec
classification below).
{ O . wdfftj'j, BxdUv'; aero genes caps uJams;
8. phlegmonic onphyxinitosac)
The bacillus was originally cultivated by Acludme
(1891) Ljut was first described in detail by Welch
and Mutts]] (1S92), who isolated it from the blood
and organs of a cadaver. This is the most important
of the clostridia causing gas gangrene. It also
produces food poisoning and necrotic enteritis in
human being* and many serious diseases in animals.
CL perfringens is a normal inhabitant nr' the
large intestines of human being* and .uni rials, ll is
found in the feces and it enntaminates (he skin of
the perineum, bu nocks and thighs.'iTe spures are
commonly found in soil, dust and air.
It is a plump. C ram positive
bacillus with straight, parallel sides and rounded
or truncated cm!*, about 4 - 6 pm * 1 pin, usually
occurring singly or in c h ain s or small bundle*, It is
pleomorphic, and filamentous and involution forms
arc common. It is capsulatcd and nnnmofilc. Spores
are central or subtcrminal but arc rarely seer in
artificial culture or in material from pathological
lesions, and their absence is one nfthe chanacteristic
morphological features of Cl. potiin gem .
It is an anaerobe
hut can also grow under m iao a u o p h ilie opndit it to*.
Oxygen is not actively toxic to the bacillus and
cultures do nut die on exposure ro air. as happens
with some fastidious anaerobes. ll grows over a
pH range of 5 .5 -8 .0 and temperature range o f
2 0 * 0 5 0 ::C. Though usually grown at 57 DC, a
C o p y rig h te d m aterial
Temperature o f 45 c'C is optimal for many strains.
The generation time at this temperature may be as
short as ten minutes. I bis property can he utilised
for obtaining pure cultures of CL perfringens.
Robertson's cooked meat broth inoculated with
rrlilures of C i pertringens and other bacteria and
incubated at 45 °C for 4 - 6 hours serves ns an
enrichment. Subcultures from this unto blood
agar plates yield pure or predominant growth of
CL pafiingcns.
Good growth occurs ] n Roberooti's cooked 1nea l
medium- T h e meat is turned pink Hut is not
digested. The culture has am acid reaction and a
sour odour.
In litmus milk, fermentation of lactose leads to
formation of acid, which is indicated by the change
in the colour o f litmus from blue to red.. T he acid
coagulates the casein (acid clot) and the clotted
milk is disrupted due to the vigorous gas
production. The paraffin plug is pushed tip and
shreds of clot are seen Clicking to the side* of the
tube, 'this is known as ’stormy fermentation.
After overnight incubation on rabbit) sheep or
human blood agat3 colonies of most strains show a
"target hemolysis’, resulting from a narrow zone of
complete hemolysis due tn theta toxin and j much
wider zone o f incomplete hemolysis due to the alpha
----------------------------------------—
Central or Cl. biferm entim
subterminal CL ^tuJfnuj]]
A B - F.
CL histolybcum
CL wrdcib
CL sparogena
O val and
terminal
Spherical
and
terminal
toxin. This double zone pattern of hemolysis may
fade on longer incubation.
Glucose, maltose,
lactose and sucrose arc ferm ented with the
production of acid and gas. Ir is indole negative,
MR positive and VP negative. H ,S is formed
iibundantly. Most strains reduce nitrates.
Spores arc usually destroyed
within five minutes bv boiling but those of the
"htod poisoning strains ofType A and certain Type
C strains reiLHt boiling for l-!i hours. Autoclaving
o f 121 :>C for 15 minutes is lethal. Spores are
resistant to the antiseptics and disinfectants in
common use.
Q p erftiftg efls strains are
class] tied mro live types, A to E hbased on the toxins,
they produce. Though the bacillus produces a latge
number cit toxins, typing depends on the four'major
toxins'- Typing is done by neutralisation tests with
specific antitoxins by intracutarieous injections in
guinea pigs or i.ntravenous injection in mice.
Cl. perfringens is one of the most prolific
ot toxin-producing bacteria, forming at least 12
distinct toxins, hesidcs many other enzymes and
biologically active soluble substance*. The four
‘major toxins-', alpha, beta, epsilon and iota, arc
predominantly responsible for pathogenicity. The
CL pertringens CS. M ax
CL .<L'prjL iJj]]
d . (Jqtn'p^i
CS. novyi CL batutinuju
CDF
CL difficile Cl. fertium G/-tvn.’hfcafTifli
C l. retarrf Cl refa™fnt>jpbum
C l tphtttoidcs
C o p y rig h te d m aterial
 * Cluslridium t 2 51
ClQllridl* « human pitfiogang yolk. The opalescence in the egg yolk media may
he produced by other lecithinase forming bacteria
A . T h e g s j gangrene group:
also (CL mivyi, CL hitcrmenTsns, some vibrios, some
]. Established CL pet ft; .ttrerii aerobic spore bearers). In the case of these bacceru.,
pathogens CJ. septreum the reaction is not neutralised by the CL pcrLr>ngcns
Cl.
antitoxin, except with CL hi ferments ns which
2. Less pathogenic Cl. hrstoh~riciim
Cl. falkx produces a serologically related lcciihlnase.
3. Doubtful pathogens C l lik n n e n rrn : The alpha toxin is hemolytic for the red cells
C l spvrogerKH of most specLCS, except horse and goat, due to its
B. Tetanus: Cl. return action on the pho&phulhiids on the erythrocyte
membranes. The ly-.is is of the hot-cold variety,
C . Food poisoning:
being best seen after incubation at 37 followed
1. Gastroentei n i-- Cl. poihngens
by chilling at 4 '3C. It is relatively hear stable and is
{Type A)
2■ Necroli-iiiK enten'i- CL priirmgrnb only partially mactuvated by boiling for five minutes.
{Type C ) Beta (J3), epsilon ($) and iota ( t > toxins have
!3. Bunilism CL buruJjVjurrr lethal and necrotising properties. Gamma (y) and
D . Acute colitis a J if c le eta (ll) toxins have minor lethal actions. The delta
(5) toxin has a lethal effect and is hemolytic for
mL]: h j.
(a ) ioxin is produced by all types of Ci. the red cell? of even-toed ungulates (sheep, goats,
y i r r i r i i and irost j In indantly hv Type A s1t :iins. pigs, catdc). The theta (0) toxin is an oxygen labile
Tbi** is the most important toxin biologically and hemolysin ant Lgcni call y related to streptolysin O­
is responsible for the profound toxemia of gas lt is also lethal and a general cytolytic toxin. The
gangrene. It lethal, dermonecmtK and hemolytic. kappa (ie) toxin is a collagenasc. The lambda (X.)
It is a phospholip idase [Lccithinasc C) which, in toxin is a proteinase and gelatinise. The mu (p)
the presence of Ca” and M g " ions, splits lecithin toxin is a bysluronula-He and the nu (v) toxin a
into phu&phoryl cholrne and d i^ ly c e n d e . This deojtylri l>oruclcasc.
reaction is seen as an opalescence in s « u m or egg Besides the toxins, Q/wr/hiJ^eiw also produces
yolk media and is specifically neutralised by the other soluble substances, some of which possess
aruitoxin. When Cl. perfringens is grown on a enzymatic properties. These include the enzymes
medium containing 6% agar, 5% Filde-- pep:ic which destroy the blood group substance, A and
digest o f sheep blood and 20% human serum, with H< a neuraminidase which destroys myxovirus
the antitoxin spread on one half of the plate. colonies receptors on red blood cells, a substance which
on the other half without the antitoxin will he renders red blood cells panagglutinable by exposing
surrounded by a zone of opacity. There w ill be no their T an Hgeos, j hemagglutinin active against the
opacity around the colonies on the half of the plate red blood cells of human beings and most animals,
with the antitoxin, due to the specific neutralisation a fibrir.olvsm, a hemolysin distinct from alpha,
of the alpha toxin. This specific letichmase effect, theta and delta tov ins, histam ine, a “buret ing factor^
known as the Nagler react iunh is a useful test lor which has a specific action on muscle tissue and
the rapid detection ui Cl. perfrin^ens in clinical may be responsible for the characteristic muscle
specimens (F ig, 2 8-1). The incorporation o f lesions in gas gangrene and a circulating factor'
neomycin sulphate in the medium makes it more which can cause an increase in the adrenaline
selective, inhibiting coliforms and aerobic spore sensitivity uf the capillary bed and also inhibit
bearers. Human serum may be replaced by 5% egg phagocytosis.

C o p y rig h te d m aterial
CL pcrfringcns producer the following human
infections:
CL pcrftingens Type A is the
predominant agent causing gas gangrene- It may
occur as the sole etiological agent hut is more
commonly seen in association with other clostridia.
as well as nondostridial anaerobes and even aerobes.
All clostridial wound infections do not result in
gas gangrene. More commonly* they lead only to
wound contamination, or anaerobic cellulitis. It is
only when muscle tissues arc invaded that gas
gangrene (anaerobic myositis) results.
Snme strains o f Type A
(food poisoning strains) can produce firod poisoning.
T hey are ch aracterised by the marked heat
resistance of their spores and the feeble production
[>f alpha and theta toxins. They have been shown
to produce a beat labile entcrotoxin which, lihe
the enterotuMins of V choJcmc and enterotoxigenic
E. c o ll leads to fluid accumulation in rhe rabbit Cl. ptrfrj’n ^ n s
ileal loop. Type A (and occasionally Type D) strains have been
Food poisoning by Cl. ptffrin gcn s is usually isolated from gangrenous appendicitis- T h e
caused by a cold or warmed up meat dish. When demonstration of antitoxin in these patients and the
contaminated meat i* cooked, the spores in the beneficial effects of the administration of antitoxin also
interior may survive. During storage or warming suggest the etiological role o f the bacillus, in this
they germinate and multiply in the anaerobic condition. It has been proposed that the toxemia and
envi ton merit in the cooked meat. Large numbers shock in some cases of intestinal obstruction and
of Clostridia may- thus be consumed* which may peritonitis mav be due to the toxin o f CL pciinngcns.
pass unharmed by the gastric acid due to the high This is a severe and
protein in the meal and reach the intestines where often fatal enteritis known by different names in
they produce the encerotoxin. After an incubation different countries: Germany (Darmbrand), New
period o f b -2 4 hours, abdominal pain, diarrhea and Guinea (pigbelj* East Africa, Thailand and Nepal.
vomiting set in. The illness is self-1 imited and It is caused by O - perfcngCits type C Strains with
recovery occurs in 24—48 hours. Diagnosis is made heat resistant spores which germ inate in the
by isolating heat resistant CL perfringens Type A intestine producing beta toxin causing mucosal
from the feces and food. A s this maybe present m necrosis. The evocative nanie'pigbei’ iB New Guinea
normal Intestines, isolation from feces, except in pidgin for abdominal pain and diarrhea following
large numbers Is not meaningful. Isolation from unaccustomed feasting on pig meat along with
food has to be attempted by direct plating on nypsm inhibitors like sweet potatoes. Immunisation
selective media, as the bacillus is present in food with type C toxoid has been shown to protect
mainly as the vegetative cells. against this condition.

C o p y rig h te d m aterial
 Major roxins Minor toxins

Typ c Pathogenicity

a f* e i Y & A e k M V

A Ga* gangrene: wound infections, stpricrmia 4-4 4- - - - - -

Food poisoning' +■■++ - - - - - 44

H-
B Lamb dysentery + ++ + +4 #4 + - 44 - 4 +4 +4 + ++

C Enteritis in animals tf+ 4 +4 - * - - S♦ H 44 + - -

Enrcntis necroticans in human beinjjs + 4 +■ ++ =■ * 4 =■ “ ~ - - 4*4

D Enterotewtemia o f sheep t 4+ - 444 - - - *4 +4 *4

E Doubtflil pathogen of sheep and Little + 4''+ - +++ - - - 44 -H- +4

 Q jit'rfringen* lu^.
beer reported to produce two rare but serums
infect ions nt the biliary tract - acute emphysernarou*
cholec-csritis a ill] postchulecYHicaouiy septicemia,
. hvaluronidase and the delta 'oxin an oxygen labile
G is gangrene of the
abdominal wall has been reported as an infrequent
complication o] abdumimJ sutgerv. The infection
is endogenous, the organism being derived from
the gut and contaminating the abdominal wall
during surgery Gas gangrene of the thi^h as a ic-suli
ot infection tracking frum the abdomen lias also
been reported.
Brain
abscess and meningitis due to CL pcrfnngcm have
been reported very rarely.
EVnophThulmLli* lIul- to CS.
perfringens has occasionally followed penetrating
eye injuries.
Clostridial infection o f
the chest cjvity may foils?w peolerrirlng WuUnJs of
tlic thorax. This is more often seen in batrlc
casualties than in civilian situations.
Infection o f the
urinary tract may occasionally follow surgical
procedure such as nephrectom y. C lostridial
infection of tht uterus is,i serious and not infrequent
condition, com m only associated wiih septic
abortion. Septicemia i-s common in this condition.
. -L .. f:»F R U ,*11 “L “ Til L ^ '\ ■
This bacillus was first described by Pasteur and
Joubcrr {1987) and called Vlbrion seprique. It is a
pleomorphic bacillus, about 3—9 pm * 0.6 pin in
si/e, forming uv:d, central nr subterminal h]"kxfCs. If
is motile bv peritrichate flagella. Growth decors
anacmhically on ordinary media. The colonies am
irregular and transparent inituUv, turning opaque
on continocL] incubation. Hemolysis occurs on
horse blood agar. Growth is promoted bv glucose,
It is saccharolytic and produces abundant gas.
Six groups have been recognised, based on
som atic and flagellar antigens. Cl. teptitruni
produces at least four distinct toxins and a
fibr irmly sin, The alpha toxin is hem olytic,
dermonecrotic and lethal, the beta toxin is a
leucatcdc deoxyribonuclease, rhe gamma toxin a
hemolysin.
CL septiaun is found in the soil or in animal
in res tines. It is associated with gas gangrene in
human brings, usually with other clostridia. It also
causes ‘bravy1 in sheep and ’malignant edema' in
cattle and sheep.
(C7, oedemaheus)
This is a large, stout, pleomorphic, Gram positive
bacillus with large, oval, subterminal spores. It is
w idely distributed in soil. It is a strict anaerobe,
readily inactivated by exposure o f cultures to air.
lour types (A to D) are recognised* based cm the
production o f toxins. Onh type A is of medical
importance, as it causes gas gangrene. Gas gangrene
caused by Cl. novyi is characterised by high
mortality and Large amount* of edema fluid with
little or no observable gas in infected tissue. Other
types produce veterinary disease,There was a lethal
outbreak of CL tioVyi rype A infection in heroin
.u ld lets in Britain in 2000-
CiLCfTilBliE
This is an acridly proteolytic clostridium, forming
oval, subterm inn I, bulging spores. T h is is
aerotolerant and some growth may occur even in
aerobic cultures. It forms at least five distinct toxins.
It is infrequently associateJ with gas gangrene- in
human*.
fr^ -r^ n if
Oakley ( 1 ^ 4 ) defined gas gangrene as n rapidly
spread Ini', edematous myonecrosis, occurring
characteristically in association with severe wounds
o f extensive muscle masses that have been
contam inated with pathogenic closfrid'ia,
particularly with CL perA ingais, T he disease has
C o p y rig h te d m aterial
 4 C l r>s1 ri di U m ►
been referred to in the past as "malignant edema'.
Other descriptive Terms that have heen used are
"anaerobic (clostridial) myn:-'i[is' and 'clostridial
myonecrosis'.
Gas gangrene is characteristically a disease of war,
in which extensive wounds with heavy comaminatiun
□re very common. In civilian life, the disease generally
follows road accidents or other types of injury
involving crusEiing of large musefe misses. Rarely, it
may even follow ^Legical operations.
The bacteriology o f gas gangrene is varied-
K a rely is this due to infection with a single
Clostridium. Generally; several species of clostridia
arc found in association with anaerobic streptococci
and facultative anaerobes such an E. cnli, pmtcun
and staphylococci. Among the pathogenic cld't i idia,
O- periringrens is the mos: frequently encountered
(approximately GO per cent}, and Cl. noyyfand Cl.
scpficum being the next common (2 0 -4 0 per cent),
and CL histoh^k-um less often. Other closeridia
usually found are Cl. sporogencs, Cl. riff fax,
Cl. hjftrtTJenruis, Cl. sordeifir, Cl. jwrtifdeTjJ'mnand
Cf. tierr.'iim. These may not be pathogen1..: by
themselves. The relative incidence of rhe different
species varies in dliferent Series o f ra&e^ and may
he a reflect inn of the distribution of the species in
different suits.
Clostridia usually enter the woundi- .lIuiic with
implanted foreign panicle such as soil (particularly
manured or culnvated soil), road dusif bus of
clothing or shrapnel. Clostridia may also be present
on the normal ski :i, cspec ially on the pc i i-icum and
thighs. Infection may at times he endogenous. Gas
gangrene may occasionally follow clean surgical
procedures (especially amputations for vascular
disease) and even injections i c-prcialk adrenaline).
The mere presence of clostridia in wounds does
not constitute gas gangrene. Mac Gen nan has
distinguished three types of anaerobic wound
infections:
Simple wound contamination with no invasion
of the underlying tissue, resulting in hide more than
some delay in wound hcaling-
255
AnaetobiL cellulite in which clostridia invade
the fascial planes, with minimal toxin pi*eduction
and no invasion of muscle rissues. The disease is
gradual in onset and may vary from a limil red Lgas
abscess’ to the extensive involvement o f a limb.
Anaerobic myositis or gas gangrene, which is
the m< nr se ri-nus, assoc i.tied with dnstri.iial i -nvaMon
of healthy muscle tissues and abundant formation
of exotoxins. Gas gangrene results o nly if the
conditions favourable for clostridial multiplication
exist in the wound. The most important of these is
low oxygen tens inn.'Hi is is achieved ideally in battle
wounds in whi. h there arc implanted bullets or
shell fragments, along with bits of clothing and
soil particles.. The ionised calcium salts and silicic
acid in the soil cause necrosis. Gnashing tissue ot
tearing of the artcriep produces anoxia o f the
muscle- Extravasai ion o f blood increases the
pressure on the capillaries, reducing the blood
supply s. Iill further. The Eh and pH o f tile damaged
tissues fall, and these changes along with the
chemical changes that occur within the damaged
and anovic muscles, including breakdown o f
carbohydrates and literati, m of aminoacids from
proteins, provide an ideal pabulum for rhe
proliferation of anaerobes. Ex ITavasated hem oglobirt
and myohe mngloh i n are reduced and cease to act
as oxygen carriers. As a result, aerobic oxidation is
halted and anaerobic reduction of pyruvate to lactate
leads to a further fell in Eh.
The clostridia multiply and elaborate toxins
which cause further tissue damage. The lecithinascs
dam age cell membrane!; and increase capillary
permeability, leading to extravasation Hn.d increased
tension in the affected muscles, causing further
anoxic dam age. T h e hem o lytic anem ia and
hemoglobinuria seen m Cl. peiinnfiens infections
arc due to the lvsis of erythrocytes by the alpha
toxin. The collagenases destroy collagen barriers
in the tissues and hyaluronidases break down the
intercellular substances further1ng invasive spread
by the clostridia. The abundant production of gas
reduces the blood supply still further by pressure
C o p y rig h te d m aterial
2S6 4 Tgxftwok o1 Microbiology ►
eBects, extending the area ofiiiw ic damage. It thus
betaines passible far the infection to spread from
the original -site, miking the lesion a progressive
one.
The incubation period may be as short as seven
horns or as long as six weeks after wounding, the
average being 1 (H -3 hours xvLth CL p£tftir\gtri£f
2 -3 days wi,th C l scpticum and 5 - 6 days with
Cl. novyi infection. The disease develops with
increasing pain, tenderness and edema of the
affected part along with systemic signs of toxemia.
There is a thin watery discharge from the wound,
which later becomes profuse and strosanguinous.
Accumulation of gas makes the tissues crepitant.
In untreated cases, the disease process extends
rapidly and inexorably. Profound toxemia and
prostration develop and death occurs due to
circulatory failure.
L a b o r a to r y diagnosis: T he diagnosis of gas
gangrene must be made primarily on clinical
grounds, and the function o f the laboratory is only
to provide con ti n i l at ion of the clinical diagnosis as
well as identification and enumeration of the
infecting organisms. Bacteriological examination
alto helps ro differentiate gas gangrene from
anaerobic streptococcal myositis, which may be
indistinguishable from it clinically in the early
stages. In the latter, Gram stained films show large
numbers of streptococci and pus cells, but not
bacilli, contrasting with the scanty pus cells and
diverse bacterial flora seen in films from gas
gangrene.
The specimens to be collected arc: (1) films
from the muscles at the edge o f the affected area,
from the tissue in the necrotic area and from
th e exudate in th e deeper jWt5 of th e wound;
(2) exudates from the pares where infection appears
to be most active and from the depth; of the wound*
t o be collected with a capillary pipette o r a swab;
and (3) necrotic tissue and muscle fragments.
G ram stained films give presumptive
information about the species of dostridia present
and their relative numbers. The presence of large
numbers of regularly shaped Gram positive bacilli
w ithout spores is strongly suggestive o f CL
perfeiiigens infection. 'Citron bodies' and boar or
leaf shaped pleomorphic bacilli with irregular
Staining suggest C l K^itJL-ujn. Large bacilli with
oval, sub terminal spores indicate C l noryi- Slender
hacilii with round, terminal spores may be CL letuii
or C l rctxnrimorphuni.
Aerobic and anaerobic cultures are made on
fresh and heated blood agar, preferably on 5 -6 per
cent agar to prevent swarming, A plate of serum
or egg yolk agar, with C l pcHr'mgcm anriitcwin
spread on one half is used for the 'Naglcr reaction.
Four tubes of Robertson's cooked meat broth are
inoculated and heared at 100 ’C fer 5, 10, 15 and
20 minutes, incubated and subculture*! on blood
agar plates after 2 4 -4 8 hours, to differentiate the
organisms with heat resistant spores. Blood cultures
arc often positive, especially in Cl. perfringens and
CL sepaom i infections. However Cl. pertringens
bacteremia may occur without gas gangrene. The
isolates are identified based on their morphological,
cultural, biochemical and toxigenic characters,
P r o p h y l a x i s am i th e r n p y : Surgery is the
most important prophylactic and therapeutic
measure in gas gangrene. All damaged tissues
should be removed promptly and the wounds
irrigated to remove blood dots, necrotic tissue and
foreign materials. In established gas gangrene,
uncompromising excision of all affected parts may
be life-saving. Where facilities exist, hyperbaric
oxygen may be beneficial in treatment.
Antibiotics are effective in prophylaxis, in
combination with surgical methods. T he drug o f
choice is metronidazole given IV before surgery
and repeated S hourly for 2 4 hours. A s mixed
aerobic and anaerobic infections are usual, a more
broadspcctru m antibiotic prophylaxis, such as a
combination of metronidazole, gentamicin and
amoxycillin is advisable.
Passive ImmurdsaLian wnh 'anti-gas gangrene
serum.' (equine polyvalent antitoxin in a dnse o f
l£>t000 IU Cl. periringens, 1 0 ,0 0 0 1U CL noyyi and
C o p y rig h te d m aterial
 . 1..L

5j,000 IU Cl. septicum antitoxin IM <]r in The optimum tcmpciatutc is G7 *C and pH 7.4. It
emergencies IVr) used to be the common practice grows on ordinary media. Growth is improved by
eh prophylaxis. However, in view of its uncertain blood and serum but not by glucose. Surface colonies
effic*ty and availability, it.H use bai become rure. are difficult to obtain an the growth has a marked
tendency to swarm over the surface o f the agar,
..... ■ " •;
especially if the medium is moist. An extremely
CL retani is the causative organism uf tetanus, fine, translucent film o r growth is produced that is
ft-ranus has heen known from very early rimes, 0 Tactically invisible, except at the delicately
h aving hee n described by Ii\ppocrates and Arcfaeus. filamentous advancing edge. This property enables
Carle and Rattone (1894) transmitted the disease the separation of Cl. teiani from mixed cultures. If
tO rahbits. N icolaier (1 9 9 4 ), studying the the water of Condensation nr the bottom of a slope
experim ental disease, suggested that the of nutrient agar is inoculated with the mixed cell
manifestations of tetanus were due to a strychninc- culture, after incubation anaerobically fbl 24 hours,
likc poison produced by the bacillus multiplying subcultures from the top of the tube will yield a
local]v. Rosenhach {1 Filth) demonstrated a dernier pure growth o f the tetanus bacillus (I'ild^s*
bacillus wifh round terminal spores in a case o f technique).
tetanus- The final proof of the etiological n>k o f ]n deep agar shake cultures, the colonies arc
the bacillus in tetanus was furnished by Kltasaro spherical fluffy balls, 1-3 mm in diameter, made
(1 8 8 9 ) who isolated it in pure culture and up of filaments with a radial arrangement, In gelatin
reproduced the disease in animals by inoculation scab cultures a fir mcc type of growth occurs, with
of pure cultures. slow liquefaction.
C l tetzni is widely distributed in soil and in the It grows well in Robertson's cooked meat broth,
intestines ofhuman beings and animals, it is ubiquitous with turbidity and some gas formation. The meat
and baa bain recovered from a wide variety o f otlier is ciot d ig e s t e d b u t is t u r n e d bl.ivk dm prolon ged
smtiers, including street and hospital du-fl:, cotton wool, incubation.
plaster nf Pans, bandages, catgut, talc, wall, plaster
and clothing. It may occur as an appartndy harmless
contaminant in wounds.
I t i) 1 Gram positive, slender
bacillus, about 4-H yim * G.5 p.m though there may V\
be considerable Variation in length. It has a straight
axis, parallel sides and rounded ends, Ir occurs singly
and occasionally in chains. The spores arc spherical,
terminal and. hulglng, giving ihe bacillus the
characteristic ‘drumstick1 appearance (Fig- 28-2)r
'Hie morphology of the spore depends on its stage
o f development and the young spore may be oval
rather than spherical. It is noncapsulated and motile
bv prntTLchiLte flagella. Young cultures, are strongly
Gram positive but older cells show variable staining
and may even be Gram negative.
It is an obligatory
anaerobe- that grows only in the absence o f oxygen.
258 i T e x t b o o k 01 M ic r o b io lo g y ►
On btoud a hcrtwlyt-]' is ~
t■ kLul'L'lI, which
larer develops into 3 hemolys-is, due to the
production of hemoly5;u:i (tctanolysir),
Bine hem ic a I re a c tio n s : Cl- reran! has feeble
proteolytic but no saccharolyrlL property. It docs
not attack any sugar It forms indole It is M R and
VT negative, H^S is not formed. Nitrates arc not
reduced. Geladn liquefaction occurs very slowly A
greenish fluorescence is produced on media
containing neutral red (as on M icC o n k e y s
medium}.
R esistan ce: The resistance of tetanus spores to
heat appears To be subjectto Strain differences. Most
arc killed by boiling for 10-1 5 minutes but some
resist bo iling for upto three hour;. When des tract ion
of spores is to be ensured, autoclaving at 121 °C
for 20 minutes is recommended. On the other hand,
when heat is applied in older to free cultures of C l
tft. in; Jn >r:i minsyvjringcontami11ants, it i- inipcsrt.ml
not to exceed 80 "C for 10 minutes as even this
mild treatment can cause considerable destruction.
Spores are able to survive in soil far years-, and are
resiscant ro most antiseptics. They are not destroyed
by 5% phenol or 0.1% mercuric chlomlle solution
io two weeks or more, lodi nc ( 1% aqueous solution)
and hydrogen peroxide {10 volumes) kill the spores
within a few hour?.
(Classification: Ten serological type* have been
recognised based on agglutination (types 1 to X).
Type VI contains nonfligellated strains. All other
types possess type specific flagellar antigens. All
the types produce the same toxin, which is
neutralised by antitoxin produced against any one
type.
Toxins: C l rerani produces at least two distinct
toxins— a hemolysin (tefanp/tr.'n) and a powerful
neurotoxin (tefanospas/njn). T h e two are
antigenically and pharmacologically distinct and
their production i-: mutually independent- A thiind
toxin, a nonspasm ogenic, peripherally active
neurotoxin, has been identified. It is not known
whether this- play? any role in the pathogenesis of
tc!:mu-i.
Tennolysin is a heat Labile, oxygen labile
hemoly-.in, anrigcnieally related to the oxygen labile
hemolysins produced by CJ. ptnirinfi&ns, Cl. tuivyi
and Str. p y ogen s. It is not relevant in the
pathogenesis of tetanus.
Tetanospasmin is the toxin resportMbl-e for
tetanus. It is oxygen stable but relatively heat labile,
being inactivated at 65 DC in five minutes. It is
plasmid coded- It get? toxoided spontaneously nr
in the presence of low co n cen tratio n s of
femmaldehyde. It is a good antigen and i? specifically
neutral^ed by the artiro*in, The toxin has been
crystallised, k is a simple protein composed o f a
single polypeptide chain, On release from the
bacillus, it is autolvsed to form a heterodimer
consisting of a heavy chan (93,000 M W ) and a
light chain (52,000 M W ) joined by a disulphide
bond. Tetanus and botulinum toxins resemble each
other irt their ammnaL'id sequences.
The purified toxin is active in extremely small
amounts and has an M LD for mice o f about 50-75
* 10 6 mg. The amount of toxin priiduccd depends
on the strain o f bacillus and the type o f culture
medium used. Its M LD for human beings is about
130 nanograms. There is considerable variation in
the susceptibility of different species of animals to
the toxin. The horse ^ the mosrsusceptiblr. Guinea
pigs, mice, goats and rabbits arc susceptible in that
descending order, birds and reptiles are highly
resistant. Frogs, which are normally insusceptible,
may be rendered susceptible nv elevating their body
temperature.
Fj hth riv e n !city : CJ. reran; ha? little invasive
power. W ished spores inlected into experimental
animals do not germinate and arc destroyed by
phagocytes. Germination and toxin production
occur only if favourable condi nona exist, such an
reduced O -R potential, devitalised tissues, foreign
bodice or concurrent infection. The toxin produced
locally is absorbed by the motor nerve ending? and
transported ro the central nervous system
intraxnnally.The tns in i; specifically and avidly fixed
by gangliosides of the grey matter of the nervous
C o p y rig h te d m aterial
 * C lD S ir dium ►
tissue. [ itanospasmm resembles strychnine in its
effects. The tetanus tyx in specifically blocks synaptic
inhibition in the spinal cord, presumably at
inhibitory terminals that use glycine and GABA ;lk
neurutransmiIters. The toxin acts presynapti .■ally,
unlike strychnine which acts postsynapthallYr The
abnhiinn of spinal inhibition causes uncontrolled
spread of impulses initiated anywhere in the central
nervous system. Tins results in muscle rigidity and
spasms due to the simultaneous contraction of
agon i-sts and antagonists, in the absence of reciprocal
inhibition. The convulsion pattern is determined
by the most powerful muscles at a giirn point, and
in most animals i-. characterised by tonic extension
of the body and of all limbs.
The toxicity of tetanospasmin is influenced by
the route by which it is administered. Given orally,
it is destroyed by the digestive enzymes and is
without effect. Subcutaneous, intramuscular and
intravenous injections are equally effective.
Intrancural injections are more lethal and injections
directly into the central nervous system very much
more so. The route of administration also modifies
the clinical picture. Experimental tetanus may
accordingly, he o f the Mocal', 'ascending' or
'descending' variety. These differences aie related
to the manner in which the toxin reaches and is
disseminated in the central nervous system.
When the toxin is inoculated intramuscularly
in f i n e i it the hindLLmhs, tonic spasms of the muscles
o f the inoculated limb appear fust. This is known
as t o o l tetanus and is due to the toxin acting on
the segment of the spinal cord cuntaini iig the motor
neurons o f the nerve supplvingthc inoculated area.
Subsequent spread of the toxin up the spinal cord
causes 'ascending tetanus'. T h e opposite hindlimb,
rmnk and forelimbs are involved in an ascending
fashion. If the toxin is injected intravenously,
spasticity develops first in the muscles of the head
and neck and then spreads downwards (descending
tetanus). This type resembles the naturally occurring
tetanus in human beings.
2Sy
TETANUS
Tetanus is characterised by tonic muscular spasms,
usually commencing at the :-he of infection and in
all but the mildest cases becoming generalised,
involving the whole of the somatic muscular system.
Most frequendy, the disease follows injury, which
may even be too trivial to be noticed. Puncture
wounds are particularly vulnerable as they favour
the growth of the anaerobic bacillus. Rarely, it may
follow surgical operations, usually due to lapses in
asepsis. Sometimes the disease maybe due to local
suppuration, such a£ Otitis media (otogenic tetanus).
Tetanus is an important complication of septic
abortion. It may be caused by unhygienic practices,
such as application of cpwdung on the umbilical
stump or rituals Such as caibofing nr circumcision.
Tetanus may also be caused by unsterile injections.
T he incubaf.Mn period is variable, from two days
to several weeks, but is commonly 6 -1 2 days. This
is influenced by several factors, such as the site and
nature o f the wound, the dose and toxigenicity o f
the contaminating organism and the immune status
o f the pat Lent. T h e incubation period ia nf
prognostic significance, the prognosis being grave
when it is short. Ol similar significance is the
interval between the first symptom of the disease,
usually trismus, and the onset of spasms (period o f
onset).
Tetanus was a serious disease with a high rate
o f mortality, SO-90 per cen t, before specific
treatment became available. Even with proper
treatment the case fatality rate varies from 15r-50
per cent. Tetanus neonatorum and uterine tetanus
have very high fatality rates (7CMQGpcr cent), white
otogenic tetanus is much less serious.
Tetanus is more common in the developing
countries, where the climate is warm, and in rural
areas where the soil is fertile and highly cultivated,
where human and animal populations are
substantial and live in close association and where
unhygienic practices are common and medical
facilities poor- In rural India, tetanus was a common
C o p y rig h te d m aterial
cause of death, particularly in the newborn. I5ui
immunisation of in Unis and expectant mi others has
reduced the incidence to a large extent.
T h e diagnosis of
ntinus should always be made on L-tinica] grounds-
Laboramry tests only help in confirmation. Not
infrequently, it may nm Ik ptwsihle to establish a
laboratory diagnosis at all.
Lab oratory diagnosis may he made by
demonstrarion of C l octant by microscopy, culture
or iiy animal inoculation. MicrosoopV is unreliable
and the demonstration ot the typical 'drumstick"
bacilli in wounds in itself is not diagnostic of
tetanus. The bacilli may be presem in some wounds
wit hunt tetanus developing, it may not also be
possible to distinguish by microsLopv between Cl.
tcfcim and morphologically similar b;Lrilli such as
CL tcOnoiTKtiphum and C l sphenoidcs. Diagnosis
by culture is more dependable. Isolation is mote
likeEv from excised bits of tissue from the necrotic
depths o f wounds than from wound swabs. "I'he
material is inoculated on one half of a blood agar
plate. Cl. istani produces a swarming growth which
may be detected on the opposite half of the plate
after 1 -2 days incubation an aerobic ally. The
material is also inoculated into three rubes or
cooked meat broth, one of which is heated to
? 0 cC lor 15 minutes, the second tor 5 minutes,
and [111- third left unhealed. The purpose of Iwaxing
for different pern win is to kill vegetative bacteria,
while leaving undamaged tetanus spores, which vary
widely in heat resistance. The cooked meat tubes
.ire incubaled it j ? "C and SubcultUred on one hall
ot blood agar plates daily for uplo four daVs. CL
tetan i may be iso I;Lred in pure culture by
subculluring Irom the swarming edge of the
growth. The incorporation o f polymyxin lif to
which clostridia are resistant, makes the medium
more selective.
For ide ntificatio ri and toxige nici tv testing, Hood
agar plates (with 4% agaj to inhibit swarming},
having tetanus antitoxin (1500 units per ml) spread
over one half of the plate are used. The Cl. frCan;
strai ns arc stab- inoculated <>" each half of the plate,
which is then incubated anaerobically for two days-
Ttudgtnic Cl. ttrjju strains show hemolysis around
the colonies., only on the half without the antitoxin.
Lysis is inhibited by the antitoxin on the other half.
This may help in identification of the culture as
Cl. tetani bur is unreliable as a test of toxigenic iry
since it indicates the production only of tetanolysin
and not necessarily of tetanospasmio, which is the
pathogen ic toxin.
Toxigemcity is best rested in animals. A two-to
four-day-old cooked meat culture (0 ,2 ml!) h
inoculated Into the root of the tail of a mouse. A
second mouse that has received the tetanus
antitoxin (1000 units) an hour earlier serves as the
control. Symptoms develop in the test anim al in
1 2 -2 4 Injury, beginning with stiffness ill the tar].
R iuid itv proceeds to the leg on the inoculated side,
the oppo* ire kg, tr uok a nd fore limbs, in that order.
T h e animal dies w ithin IWo davs Imt may lie killed
earlier as the appearance o f ascending rctanus is
diagnostic.
Tctanus is a preventable disease.
As rhe spores arc ubiquitous, wound contamination
is unavoidable. The disease is due to the action of
the toxin. T herefore the obvious and m ost
dependable method ol prevention Li to build up
antitoxic immunity by active immunisation by
routine immunisation o f children and booster doses
where appropriate.
The nature of prophylaxis depends Largely on
the type of the wound and the immune status
Of the patient. T h e available m ethods ot
prophylaxis are (1) surgical attention; (2) antibiotics;
and {!) immunisation— passive, active or combined.
huigical prophylaxis aims Jt removal of foreign
bodies, necrotic tissue and blood clots, to prevent
an anaerobic environment favourable for rhe
tetanus bacillus. The extent of surgical treatment
may vary from simple cleansing to radical excision,
depending on the type of wound.
Antibiotic prophylaxis aims at destroying or
inhibiting tetanus bacilli and pyogenic hacteria in
C o p y rig h te d m aterial
 * Clostridium *
wmainds £« thit the productiu11 ■iftnxml is pre\ l'Tite. 1.
In experimentally infected animals tetanus can be
preveii ted by antibiotics- when adnum^tered tour
hours after : nfcct:i)n but not alter eight hours. This
emphasises the need for prompt administration ol
antibiotics. Long anting penicillin injection is the
drug o f choice. An alternative is erythromycin 500
mg b,d, for five days. Antibiotics arc to be starred
before Wound Toilet. Bacitracin or neomycin may
be applied locally also. Antibiotics have no action
on the toxin. Hence, antibiotic prophylaxis does
not replace immunisation but serves as a useful
adjunct.
Passive immunisation is by injection of tetanus
an titoxin. A utitetanus serum ( A i d ) from
hvpci ii .1 mune horses was the preparation originally
used. The dose employed was 1 5 0 0 1U given
subcutaneously or intramuscularly in non immune
persons slum after receiving any tetanus prone injury.
ATS was useful not only in reducing the incidence
of tetanus but also in prolonging the incubalion
period and reducing the mortality when it did not
prevent the disease. However; equine ATS carried
two disadvantages implicit in rhe use of any
heterologous serum - ‘immune elimination' and
hypersensitivity; The hall-life of ATS in human
beings is normally about seven days but in persons
who have had prior injections of horse scram, .1 is
cl imi nated much more qu ickly by combi nation with
pie -existing ant iItodies. Pi tor sensitisat ion al so lends
to hypersensitivity reactions which may range from
mild Local reactions To serum sickness* and even
fatal anaphyljuds. It is, therefore, obligatory that a
rest for hypersensitivity should invariably be made
hetbro administration of ATS, The intradermj] test
for hypersensitivity, which is in common use, has
been reported to be unreliable, A 'trial' dose given
subcutaneously would be a be tier index o f
hypersen sitiv-tv. A dose of CL5 ml of ATS is given
subcutaneously and the patient observed for at least
half an hour for general reactions. As even this
dose mjiv precipitate anaphylaxis in some eases, a
syrin ge iv a d td w ith adrena !.<i. r (1 /1 0 0 0 ) rttuSt b e
261
kept ready. In persons with a histoni ot any allergy,
the trial dose should be 0.05 ml of a 1/10 dilution
o f ATS
Bovine and ovine ATS were introduced to
overcome reacrions to horse serum but these in turn
can also produce hypersensitivity. Passive immunity
without risk of hypersensitivity'call be obtained by
the use of human anti tetanus immunoglobulin
(TIG ). This in effective in smaller dose!; (250 units;)
and has a longer half-lile (3~ 5 weeks). As T IG is
prepared by immunisation of human volunteers, its
availability :s limited-
Passive immui iisari on is a n emergency procedure
to be used only once. The former practice of persons
receiving ATS every lime thev are wounded was
not only useless and wastcftil hut also positively'
dangerous. It is better to eliminate the use of ATS
altogether, tetanus being controlled by active
immunisation, with human I 1G being reserved for
emergency use in the noni immune.
Active immunisation is r o t only the roost
effective method of prophyLairi- but also the only
means whereby tetanus tbUnwi ng unnoticed irtjuri; s
can be prevented. This is achieved by spaced
injections of formed toxoid, which is available either
as 'plain toxn'd', or adsorbed o r aluminium
hydroxide or phosphate. Hie adsorbed lOXoid is a
better anhgen. The tetanus toxoid is given either
alone or along with the diphtheria toxoid and the
p e r m s v a c c in e as the ‘triple vaccine', in which
pertussis vaccine acts as art adjuvant also. A course
of immunisation consists of three doses of tetanus
toxoi.l given intramuscularly, with an interval of
4 -6 weeks between the first two injections and the
th ird dose fi m onths later (or as per
recommendations of the National Immunisation
Programme). A full course of immunisation confers
immunity for a period of at least ten years, A ^booster
dose‘ of toMOM: is recommended alter ten vears. ATS
or T [G should not hr given to an iiumunL--ed
individual. Instead, s booster dose of toxoid i- giver
if wounding occurs three vears or more alter the
full course of immuiusation. Too frequent injection
C o p y rig h te d m aterial
of toxoid should he avoided as hypersensitivity However, because the patients are isolated, there ]£
iticnonc may occur occasionally. a com mon impression that they arc highly
An illustration o f rhe efficacy o f active infectious. This is not true. TelitUis patients are
imrrtLjcitation is that in World War II, o nlv 1 2 c ascs hardly ever infectious, and person to person
o f tetanus occurred in 2 , 7J?4,S 1 y hospital transmission does not occur at all.
admissions for wounds or injuries, among the Treatment consists of enuring quiet, controlling
Ameririm soldiers who had been previously spasms, miuntaining airway by tracheostomy with
immunised. intermittent positive pressure respiration and
Com bined im m unisation consists o f attention to feeding. Human T lG 1 0 ,0 0 0 LU
administering h> a runimmuine person exposed 10 suitably diluted may be given by slow IV infusion,
the risk of tetanus T IC injection at one site, along followed, if reeded, by another 5,000 IU later. Even
with the first dose of toxoid ar the contralateral though TIG may not neutralise the toxin already
site, followed hy the second and third do sen of bound to the nervous tissue, it can inacrivate the
toxoid ac monthly intervals. Jr ts important to use unbound toxin and any further toxin that may be
adsorbed toxoid as the immure response to plain produced. Antibacterial therapy with penicillin or
toxoid maybe inhibited by T IG , Ideally, combined metronidazole should he started at once and
immunisation should he employed whenever continued for a week or more. ATS used to he given
passive immunisation is called for. intravenously in in arrive doses as part of the
Table 2 ? 3 shows the recommended integrated treatment. Several controlled trials have heen
prophylaxis of tetanus following injury. undertaken to assess the value of antitoxin and the
Tetanus patients should be treated optimum dose. The results indicate that antitoxin
in hospitals, preferably in special units. The reason is o f value in treatm ent but that 1 0 ,0 0 0 1U
for isolating them is to protect them from noise intravenously gives an gtntfl KSUltS an much higher
and light which may provoke the convulsions. doses.

Clean (wound toilet performed within six hours) Toxoid * 1 * Toxmd * 1 Toxoid * 3
Contaminated (soil or other foreign ot
necrotic material present} Toxoid * l * Toxoid x I Toxoid * 3
TIG TIG
antibiotic* antibiotics
Infected Toxoid * 1 * Toxoid * 1 Tuxuid x 3
Antibiotics TIG TIC
antibiotics antibiotics

Mote: Immune - .Patient has had a full course of three injections of toxoid.
Partially immune - Patient has bad two injections of toxoid.
M y t L i n i i i i i m L : - P a t i e n t f i j s h i d O r t * 0 1 ii
0 0 i n j e c t i o n i> F T i u t u id , O f i m c m i f t i s a r i o n s i a r u s I M l I m o w n .

T lfl - Tetanus Immune Globulin.

* The toxoid needs to be given only if three yeara or more have elapsed after active immunisation or
the last booster injection.

C o p y rig h te d m aterial

Patients recovering front tetanus should receive
:l full course of active immunisation, as an attack of
the disease does not confer Immunity. Second
attacks o f tetanus have been recorded.
Cl. fwmfrniijn causes botulism, a paralytic disease
usually presenting as a form of food poisoning.
T h e name botulism is derived from ‘sausage*
{botuhis, Latin for sausage J formerly associated with
this type of food poisonLog. Cl- iSoru/jJTum was first
isolated by van Ermertgem (189fr) from a piece of
ham that caused an outbreak of botulism. The
bacillus lSa Widely distributed saprophvte, occurring
in virgin soil, vegetables, bay, silage, animal manure
and sea mud-
It is a Gram positive bacillus
about 5 |lni x 1 Jim, noncapsulated, motile by
peritrichace flagella, producing -ubn/mmiul, oval,
bulging spores.
It is a strict
anaerobe. Optimum temperature is 35 "C but some
strains may grow even at 1-5 °C. Good growth
occurs on ordinary media. Surface colonics are large,
irregular, semitransparent, with fimbriate border.
Biochemical reactions vary in different types, Spores
are produced consistently when grown in alkaline
glucose gelatin media at 2 0 -2 5 "C. They art not
usually produced at higher temperatuies.
bpores are heat and radiation
resistant, surviving several hours at 100 C and for
upto 10 minutes at 120 aC. Spores of nonproteolytic
types of B, E and F are much less resistant to heat.
Eight types o f Cl. hotuimum
have been identified (Types A* ft, C l , C2, D, E, F,
G) based o a the immunological difference in the
toxins produced by them. The toxins produced by
the different types are identical in their
pharmacological activity but arc neutralised only
by the homologous antiserum. An exception is C2
toxin, which shows enterotosLc activity, while all
the others are neurotoxins.
CL fwmJjmpm produces a powerful exotoxin
I
that is responsible for its pathogenicity. The toxin
differs from other exotoxins in that it is not
released during the life of die organism. It is
produced intfacellulariv and appears in the medium
only on the death and autnlvsis of the cell. It is
believed to he synthesised initially a npntnxic
protoxin or progenitor toxin. Trypsin and other
proteolytic enzymes activate progenitor toxin to
active toxin.
The toxin has been isolated as a pure crystalline
protein which is probably the most toxic substance
known. Jr has a M W 70.000 and a lethal dose for
mice of 0.00 0 ,0 0 0 ,0 3 3 mg. The lethal dose for
human beings is probably 1-2 |Jg. It is a neur-otoxin
and acts slowly, taking several hours bo kill.
The toxin is relatively stable, being inactivated
only after 30^40 minutes at SO QC and 10 minutes
at 100 : C. Food suspected to be contaminated with
botulinum toxin can be rendered completely safe
by pressure cooking or boiling for 20 minutes. It
resists digestion and is absorbed through the small
intestines in active form. It acts by blocking the
production or release of acetylcholine at the synapses
and neuromuscular junctions. The onset is marked
by diplopia, dyspllagia and dysarthria due to cranial
nerve involvement. A symmetric descending
paralysis is rhe characteristic pattern, ending in
death by respiratory paralysis.
A small quantity of CL horniimmi type A toxin
injected into a muscle selectively weakens it by
blocking the release o f acetylcholine at the
neuromuscular junction. Muscles so injected atrophy
hut recover in 2 - 4 months as new terminal axon
sprouts form and restore transm ission.
Intramuscular injection of the toxin, first used to
treat strabismus, is now recognised as a safe and
effective sym ptom atic therapy for many
neuromuscular diseases.
The botulinum toxin can be toxoided. It is
specifically neutralised by its antitoxin and is a good
antigen. The tnx ins produced by the differen t type*
of C l fjofufijium appear to be identical,,, except for
immunological differences. Toxin production
C o p y rig h te d m aterial
 26 4 i Teii&ook ol Microbiof&gy *
appears to be determined by the presence of
bacteriophages, at least in types C and D.
Pathogenicity: Cl. hotulinum is noninvasive and
virtually noninfectious. Its pathogenicity' i due to
the atrion of its toxin, the manifestations of which
are collectively called botulism. Botulism is of three
types - foodborne botulism, wound botulism and
infant botulism.
FtMxlkttme hotuiixni is due It? the ingestion of
preformed toxin. The types ot the bacillus and the
nature of the food responsible vary in different
regions. Human disease is usually caused by types
A, B, E and very rarely I\ h p e s C and D are usually
associated with outbreaks in catdc and wild fowl.
Type G has been associated with sudden death in a
few patients. The source o f botulism is usually
preserved lood— meat and meat products in Europe,
canned vegetables in America and fish in Japan.
I vpc I'] is associated with fish and other seafoods-
Proteolytic varieties of CL bondimim can digest
food, which then appears spoiled. The cans are
often in Hated and show bubbles on opening.
Nonprorcolytic varieties leave food unchanged.
Symptoms begin usually 1 2 -3 6 hours after
ingestion of food. Vomiting, thirst, constipation,
ocular paresis, difficulty in swallowing, speaking
and breath i.ngcunslitute the common features. Coma
or delirium may supervene. Death is due to
respiratory failure and incurs days after onset.
Case fatality varies from 25—70 per cent.
IVouimJ boru/.'sjT] is a very rare condition
resulting from wound infection with C7. botuirmim,
FuA: cl is produced it the site of infection and is
absorbed. J lie symptoms are those of foodbnmt
botulism except tor the gastrointestinal components
which are absent. Type A has been responsible for
most of the cases studied.
botulism was recognised as a dimes]
entity in 1976. This is a to xico -infection. CL
bow lin u m spores arc ingested in food, get
established in the gut and there produce the to\in.
Cases occur in infants helow nix months. Older
child ten arid adults arc not susceptible. The
manifestations are Cfinstipation, poor feeding,
lethargy, weakness, pooled oral secretions, weak or
altered cry, flop pi ness and loss of head control.
Piri ents excrete tax in and sporet- in the r feces. Toxin
is not g e n e ra lly dem onstrable in blood.
Management consists of supportive care mid as'-isted
feeding. Antitoxins ;tnd antibiotics art not intheated.
Degrees ol severity vary from veiy mild illness to
fatal disease. Some eases of sudden infant death
syndrome have been found to be due to infant
botulism. Honey has been incriminated as a likely
fond Hem through which the bacillus enters the
gut.
L a b o r a t o r y d ia g n o s is : D iagnosis may be
ennfinned hy demonstration of the bacillus or the
toxin in food or feces. Gram positive sparing bacilli
may he demonstrable m smears made from the (bod.
CL botv.'.'num may be isolated from the food or
the patienri feces. Tire food is macerated in sterile
sal 11 re, and the filtrate '.i i:iculxteti intn nlice or gu:uca
piips intraperiHoneally. Control animals protected bv
polyvalent antitoxin remain healthy IVyiing is done
by passive protection with type specific antitoxin.
The toxin may occaslanally he demonstrable in the
parlcnPs blood, or in the liver postmortem.
A retrospective diagnosis may be made hy
detection of antitoxin in the patient's scrum but it
may not be seen In all cases.
C o n tr o l: As m ost cases of botulism follow
consumption of inadequately canned or preserved
food, control can be achieved by proper canning
and preservation. W hen an outbreak occurs, a
prophylactic dose of antitoxin should be given
intramuscularly to all who consumed rhe article of
food.
Active immunisation has been shown to he
effec:ive. 11 immunisation Is. needed, a-s in labt>rat<?iy
workers exposed to the two injections of
aluminium sulphate adsorbed toxoid may be given
at an interval of ten weeks, followed by a booster a
year later Anrhoxin may be cried for treatment.
Polyvalent antiserum to types A, B and E may be
adm in iStered as soon ass din ical diagnosis is made.
C o p y rig h te d m aterial
Supportive therapy wtth rnj.LElTena.nct: of refutation
is of equal or greater importance.
«£i;^M^)iME3' t j p m L E m ® j&wnfc&Tic
Ci. difficile wus tirnt isolated in 19.5.S from the ffccs
o f newborn infant*-. It WU ho ruined because of the
unusual difficulty in isolating it. It in a long, slender,
Gram positive bacillus with a pronounced tendency
to lose its Gram reaction, Spores are large. oval
and terminal. It is nonhemolytic, sjiccharolytie and
weakly proteolytic. It was not considered
pathogenic till 1977 when it was found to be
responsible tor antibiotic associated colitis.
Acute colitis, with of without membrane
formation, is an important complication of oral
antibiotic therapy Mans antibiotics have been
incriminated including ampteiUin, tetracycline and
chloramphenicol but Lmcamycin and clindamycin ate
particularly prone to cause pseuElomembranouH colitis.
It has now been shown that antibiotic associated
colitis in due to the active multiplication o f C l
difficile and its production o f an enterorosin as well
as a cytotoxin. D iagnosis can be made by
demonstrating the toxin in the feces of patients hy
its characteristic effect on Hcp-2and human diploid
cell cultures or by E L JS A .T h c toxin is specifically
neutralised by the O . sordWIi antitoxin. CL difficihc
Cfln also be grown from [he feces of patients.
CL difficile strains arc usually resistant to most
antibiotics. Metfftnidaiole is [he drug -of choice.
Vancomycin and bacitracin are also useful.

BprtlettlG. 1994. Clostridium diflieik. Clin Infect Dis, 18:5265.

Brazier JS. 1995. L ib an to iy d iip ou cfC L difficile associated disease. iicr Med AfjfcnafcfoJ 6:236.
Duerden BI and BS Dra-^ar (ede). 1991. Anaerobes jo human diseise. London: Arnold,
HathavR CL, 1995, Botulism, Cuit Topics Microbiol Immunol, 195:55,
Sikurai J, 1995. Timm of Cl, Perfeingens, Rev Med MicrobioL 6:175.
Sanford JP 1995. Tetanos - forgotten, but not pone. New Engl j Med, 352:812.

C o p y rig h te d m aterial
 Nonsporing Anaerobes

Anaerobic bacteria have been known since the L Cocci

original observation of Pasteur that bacteria which A. Gram positive
pfoducc butyric acid, his Vihriim bulrriyue, were a. Rphtttreptovovcus
rendered nonmotile on exposure to air (1563), hr I^ptOCOCCMS
Though many anaerobic bacteria may be B„ Gram, negative
pathogenic for human beings, EKt:v are generally YeilloncUa
neglected in diagnostic laboratories. This neglect II. Bacilli
is not because they arc uncommon, Indeed, they 1. h-tidospore forming
outnumber aerobic bacteria in many habitats, Clostridia
including most sires o f the human or animal 2. Nonsporing
bridy. Even in such seemingly aerobic situations, A. Gram positive
as the mouth and the shin, anaerobic bacteria are a, Eubacterium
ten to thirty times more frequent titan aerobes. In b. Propicmibicterium
the human intestines, they outnumber aerobic l- Lactobacillus

bacteria a thousandfold. The numbers of anaerobes d. Mobiluncus

present have been estimated to be 10* - 107ml in e. Bifidobacterium
the small intestine. ItfVm! in saliva and 10IL/g in f Actinomyces
the cnlon. B . G ram negative
Anaerobic bacteria differ widely in the degree a. Hacteroides
o f anaerobiosis required for their growth. Some b, Prevotellla
species fail to grow if the atmosphere contains as c, Porphyromonas
lirrle as 0.Q3 per cent oxygen, while at the other d. Fusobacterium
extreme, some arc acrotolcrant and may grow c. Lcptotrichia
sparsely on the surface of aerobic places. IIL Spirochetes
Consequently the techniques employed for the a. Treponema
propagation and study o f anaerobes vary in b. Bomelia
complexity. Besides the medically important species listed
Early methods of classification used such above, there are several anaerobes that occur in soil
unstable criteria as cell and colony morphology, and w ater and w hich may be o f industrial and
biochemical reactions and antibiotic sensitivity agricultural im p o rta n c e (fo r ex am p le,
patterns. Current dassifLiiation is based on DNA methanjobacteria, butyrivibrios).
base composition and analysis of the fatty avid end
products of metabolism. Medically important
anaerobes may be broadly classified as follows: A n aerob ic coccu represent a heterogeneous

C o p y rig h te d m aterial
 4 Nonsporing Anaerobes ►
collection o f cocci whose classificatlofl and
nom enclature have undergone several
modifications- They can be divided into Gram
posiir.vc and Gram negative gnoups-
A n aerobic Gram positive cocci had been
classified into the genera ftpfosrrepfoajccus and
/Vprococcus originally, based on morphology,
chain-form ing and paired cocci placed m the
former and cluster-forming cocci in the latter.
However, DNA base ratio studies have led to most
of the species formerly considered as peprococei
being reclassified as pcprostrcptocncci- They are
cocci of small size (0 .2 -2 .5 pm). Many of them
are aeratoLerant and grow well under ltfitj C O . in
an aerob.iL atmosphere.
They are normal inhabitants of rhe vagina,
in test'lies and mouth. They may cause several
clinical infections such as puerperal sepsis and other
genital infections, wound infections, gangrenous
.Lpp*: rn.Iil:i . nrm.Lrv tr.u i infections, osteomyelitis
and abscesses in the brain, lungs and other internal
organs. They are often seen in large numbers in
pus from suppurative lesions, so a Gram stained
smear may be helpful in diagnosis, Infections are
usually mixed, the cocci being present along With
clostridia or anaerobic Gram negative batilLi.
ftpruirrep rocoecu s m jerubjus is most often
responsible for puerperal sepsis and Fsr. magnus
for abscesses. Ftf. isacch-iioIyticust Pst. tetrad.'us
and J^f. prevoti air some other species commonly
present in clinical specimens.
Vcilloncllsc are Gram negative cocci of varying
s iHes occurn ng as ci iplococci, short chains Or groups.
They are normal inhabitants of the mouth, intestinal
and genital tracts. Veillunella p a m iii has been
reported from clinical specimens but its pathogeni.C
role is uncertain.
All anaerobic cocci are generally sensitive to
pen ic iLun. chloramphen icol and metronidazole, and
resistant to streptomycin and gentamicin.
A naerobic G ram P ositive B acilli
This group contains many genera, of which the
2fi7
medically re Levant E u bactcriu m ,
are
Propicni bacterium, LacfobsciMu*, Mohiluncus and
B ifidu bactti rum. O ther genera i.n this group,
AcrinoiTTvces
F and
'
AracJuiia,
- J
are dealt v ith
elsewhere.
Members of the genus EubaLlcn urn arc Strictly
anaerobic and grow very slowly. They are members
of the normal mouth and intestinal flora. Some
species (£ . brachy £ r tjmitium, E. nods rum) are
commonly seen in periodontitis- E . hn tu m is
commoitly isolated from nonoral clinical spe< imcns.
Aupinnilurvriuii) is constantly present on the
sk:-n, £ acncs is a common contaminant Lu blood
and C S F cultures.
Laciubariflus is present in the mouth, intestines
and, typically, in the adult vagina (Dodcrlcin's
bacilli). It is generally nonpathogeniC, though L
catejTjforme has been assoc i.Lted with
bronchopulmonary infections.
B i f i d o b a c r e r i i i m is a pleomorphic rod that
hIiowf true and false branching. It is present in large
numbers in the intestines and in the mouth.
jV/ob,ii'uncuf species ate m otile, curved,
anaerobic bacilli that may appear as Gmm variable
rods. M. mufieris and M . rurfj'aj have been isolated
from the vagina in bacterial vaginosis, along with
G ardncrella viginalis. iiacteri.i] vaginosis Ls a
polymicrobial infection characterised by a thm
malodorous vaginal discharge. Its 'rotten fish' smell
is accentuated by mixing it with a drop o f KOH
solution- The vaginal pH ls less than 4.5. Clue cells
(epitheLi.d cells with surface covered by adherent
baeilb) are seen in stoned or unstained film*.
A naerobic G ram N egative
B a c illi
Medically important anaerobic Gram negative
bacilli belong ro the family iJacterwfeceacand are
classified into the genera B a ctew id c s,
Fusofjacten'ufn and Lepfotrichia,
B sctcroid a ate the most common anaerobes
isolated from clinical specim ens. They are
nonsporing, nor motile, strict anaerobes, usually very
C o p y rig h te d m aterial
268 i Textbook of Microbiology *

|i|l-ii n it-]’ I■_K", jppejung an ■Hlcrt.JcT' rod.*, branching and other abdominal infections in animals and less
forms or ooceobseilli, seen F-ifigly, in or in often in humans.
short chains. They grow well on media such as- The genus Lcplotnchia coma ills the -mglv
brain, heart infusion agar in an anaerobic atmosphere species, fruccflits which was formerly known as
containing 10% C O ,. T hey possess capsular Vincent's fusiform bacillus or Fusobacfertutn
polysaccharides which appear to He virulence fusiforme. Fhey .ue long, str.'.ighl or slightly curved
factors, and antibodies to them can he detected in rods, often with pointed ends. Thev are part of the
patients. Thev are normal inhabitants of the normal oral flora and are seen in acute nccrotising
intesri.nalh respiratory and female genual tracts. lesions in the mouth.
Bactcro-des species have been classified based A common condition is Vincent's angina, which
on their sacch arolytic effects. Asaccharolytic may resemble diphtheria, with the inflamed
pigmented species have been separated as the genus pharyngeal mucosa showing a greyish membrane
PbiphyromoiuH, containing Pglngivalla respi jrisible which peels easily. Stained smears show large
for periodontal disease, F cjidodo/ifaJj-s causing fusiform and spiral bacilli.
denta] root canal infections and other species.
Moderately sacchuolvlic aperies minified by 20% A n a e r o b ic I n f e c t io n s
tile are placed in the genus Prcvotella. containing The re has been n reawakening o f interest in
F. mehninogenicM, P. buecalis, F. dcnticola and anacrotic infections during recent years. This is
Others. The genus Ratten udes proper now includes due to the availability nl improved and simplified
the important species B- frvplis, and others such techniques for the isolation and identification of
as fl, Yiilfinitiw, B. rssoHfs and B. anaerobes.
fhef:i iotaumienm. Anaerobic infections arc usually endogenous and
B. f'ragiti s is the m ost frequent of theare caused by tissue invasion by bacteria normally
nonsporing anaerobes isolated from clinical nescient <m the rasped ive bodv surfaces. Anaerobic
specimens, It is often recovered from blood, bacteria arc normally present on the skin, mouth,
pleural and peritoneal fluids, CSF, hi .ini abscesses, nasopharynx and upper respiratory tract, intestines
wounds and urogcm tal infections- P and vagina {Table 2 9 .1). Anacmhic infections
Tntkninotfeihiii is easy to recognise because of generally follow some precipitating factor such as
the black or brown colour of the colonies. The trauma, tissue necrosis, impaired circulation,
colour is not due to the melanin pigment as was hematoma formation or the presence of foreign
once thought but to a hemin derivatve. It has been bodies. Diabetes, malnutrition, malignancy or
isolated from various infections itcludnig lung or prolonged treatm ent with am inoglycoside
liver abscess, mastoiditis, intestinal le-iors and suitib^>ti.s may act as predisposing factors.
lesions of the mouth and gums- Cultures o f F- Anaerobic infections arc typically polymicrobial,
rne/amrto^euh'a and even dressings from wounds more than one anaerobe being responsible besides
infected with the bacillus give a characierisric red aerobic bacteria. W hile the infection is usually
fluorescence when exposed to ultraviolet light. localised, general dissemination iii.lv occur hv
The genus Fusohwcterium contains long, thin bactcrem::i. Tabic 29.2 lists the common sites and
or spindle shaped bacilli with pointed ends. /■. type o f anaerobic infections and the bacteria
nuefejturn is a normal inhabitant of the mouth and responsible.
is found in oral infection and pleuropulmonary JlTiere are some clinical features which suggest
sepsis. F, rteerophorum produces a wide range of the presence of anaerobie infer i ion. Pus produced
exotuAins and has been responsible l:ot liver abscess by anaerobes is characteristically putrid, with a

C o p y rig h te d m aterial

Clostridium
Actinomyces
bifidobacterium
Propionibactniiun *+
B tctsroitia frag&i
P mclttiiinogefiica
KuBobacterium
Gram positive cocci
Gram negative cocci
Spirochetes
pervasive, nauseating odour. However, there may
he exceptions; infections solely dec to B frsgilis
may be t ree of this smell. Pronounced cellulitis is a
common feature of anaerobic wound infections.
Toxemia and fever are not marked.
As anaerobes lorm
part of the normal flora o f the skin and mucous
surfaces, their isolation from specimens has to be
interpreted cautiously. The mere presence o f an
anaerobe dots not prove its causal role, Specimens
should be collected in such a manner as tn avoid
resident flora. For example, the sputum is
unsatisfactory' for culture from a suspected case of
lung abscess; only material collected by aspiration
would he acceptable.
As some anaerobes die on exposure to oxygen,
cate should be exercised to minimise contact with
air during collection, transport anil handling of
specimens. A satisfactory method o f collection is
to aspirate the specimen into an airtight syringe,
plunge the needle into a sterile robber cork to seal
it and send it immediately to the laboratory, Pus
and other fluids may be collected in small bottles
wi th airt ight caps and transported qu ickly, cn suri ng
that the specimens fill the bottles completely. Swabs
are generally unsatisfactory hut where they arc to
j? 6 f i.':'J rh r- .Ij'i.m. i - |r r H;.
+4
*
4 ++ t
++
+■ 4 4 +4
*4 4
4- + 4+ 44
+■ 4 4 4-4
+
be used, they should be sent in frtuarr’s transport
mcdiutn-
In the Laboratory, exposure should be limited
to Hu- minimum. Examination of a Gram Stained
smear is useful. Pds in anaerobic infection usually
shows a Large variety of different organisms and
numerous pus cells. Rarely, as in brain abscess, a
single type o f organism alone may be seen.
Examination of the specimen under ultraviolet
light may show the bright red fluorescence o f
P mel&ninagcniai. Gas: liquid chromatograph*1 of
the specimen mav yield presumptive information
on the types of anaerobes presen r.
Several special media have been described for
anaerobes, but for routine diagnostic work, freshly
prepared blood agar with neomycin, yeast extract,
h erein and vitam in K is adequ ate. Plates arc
inCUb&tcd at 3 7 *C in an anaerobic jar; with |Q%
C O ,. The Caspak system provides a convenient
method o f routine anaerobic cultures. Plates are
examined after 24 or 4fl houit Some anaerobes, ScLdh
as fusobactcria, require- longer periods of incubation.
[Valid aerobic cultures should always be set up. This
is necessary as a control frvr the growth on anaemhie
plates and also because in most anaerobic infections
aerobic bacteria are also involved.
C o p y rig h te d m aterial
 Central affirm:
Brain abscess B. fragilisi i^ptostrcptinroixus
Rat, fwml-, rhroar
Chronic sinusitis, otitis media,
mastoid iris, urbuid ccllulir-s I n--:'bacteria (aerobes frequently responsible)
M outh and jaw.
Ulcerative gingivitis (Vincent's) Fusobictcria, spirochete*
Dental abscess, cellulitis; Mouth anaerobes,
Abscess and sinus of jaw AttnuAiyttt, other mouth anaerobes.
Reepira t£>ry.
Aspiration pneumonia, knit; abscess, Fwubacteria, P- melstniriogeftrc*,
bfflnehieaaais, empyema anaerobic cocci;. B. fiagilis rarely
A bdom in al.
Subphreni-c, hepatic abscess;
appendicitis, peritonitis;
ischiorpcraL -bsLl^s. B. iragilis
wound Infection after
colorectal surgery
Female genirxlia:
Wound infertiun hrlluwiug
genital surgery;
Puerperal sepSii;. P. me/artinogtJUL’d,
tubo-ovarian nbscesi, h i aerobic cocci; li. ftngilis
Bartholin's HbSf«ssn
septic abortion Cefliral anaerobes and CL pairipgcw
Stitt jjj J soft tissue
Infected sebaejous cyst. Anaerobic cocci
Breast abscess, axillary abscess Anaerobic cocci; RmelxniDQgznfca (Staph. aureu*
commonest cause)
Cellulitis, diabetic ulcer, gangrene ft, iragilh and ■'fliers.

DiKiden D L uid QS Dr*pjr (cds)- 1991- Ajiacrtahes jji Hiimtn Dbea*tr London: Arnold,
hintgold M, I99J. Overview ofclinL-caltv important anaerobes. Clin Intecr Dts, 30: &205.
Kasper E3l.. 1993. Infections due to mixed anaerobicor^Jiusms. In Harrison's Principles o f Internal Medimne, 14 edn, New
York; McGraw K ill

C o p y rig h te d m aterial
 Enterobacteriaceae I: Conforms * Proteus
The predominant aerobic bacterial flora of the i.irj^
i met-tines of human be ings and an ina.i la is -composed
uf nonspurmg, nonarid fast, Gram rutj^lLYe bacilli.
1’ liry exhibit general morphologi cal and
biochemical similaritKs and aie grouped together
in the large and complex family Eniejviucferjacede.
M em bers o f this family may or may nor be
capsulared and are motile by periirichale flagelk,
nr arc nor motile. They arc acmhiv and facultatively
anaerobic grow readily in ordinary' media, ferment
glucose producing a^ id and gas or acid only, reduce
nitrates to nitrites and form catalase but not oxidase.
W ith in the family, rhey exhibit very wide
biochem Leal and antigenic heterogeneity- Though
the family j s .tbdivided into groups or tribes, genera,
subgenera, species and types, many strains are met
with that possess every conceivable combi nation
of characters and do not fall into any such arbitrary
taxonomic category. The Frequency of genetic
mechanisms such as conjugation and transduction
in these bacteria contributes to their infinite variety,
Classificaii'm of these bacteria into well demarcated
compartments, Though necessary for their systematic
s tu d y , would therefore be artificial. T h e
classification o f E n tcm hacti-riaccae has hcen
controversial and there have been successive changes
in their grouping and nomenclature.
The oldest method was to classify these bacteria
into three groups based on their action on lactose.
T. Lactose fermenteis (for example
Escherichia,
Klebsiella)
II. Late lactose fermenters (for example
Shigella sonnei
‘Paracolons1)
III. NonlactoSe fermenters (for example
Salm onella,
Shigella)
This method of classification was derived from
the use of lactose in MacC on key’s medium, the
most popular medium for the isolation of feral
bacilli. Though taxortOTmeally unacceptable, this
scheme had practical value In diagnostic
bacteriology. The majority o f the commensal
intestinal bacilli arc lactose fermenting (L F ). As
the most common member of this group is the colon
bacillus, or Escherichia coh. all lactose Fermenting,
enteric ban Hi were called colifbrm bacilli. The
major intestinal pathogens, Salmonella and Shigella
are nonlactose fermenters (N LF), and hence readily
detectahle by the colourless colonies they form on
MacConkeyfs medium. There remaned a small
group which showed delayed fermentation o f
lactose and with the exception of 5higr.Ua sonnei,
they were all commcn^ils.This heterogenous group
o f late lactose fermenters was called pamco/on
batiUi.
Classification based on a single property, such
as lactose fermentation, is contrary to modern
taxonomical concepts. The current practice is to
group together bacteria that possess a number o f
com m on mo rpho logical and biochem ical
properties, and similar D N A base compositions.
W hile the three widely used systems for the
classification o f /vnrernbserfer/aceae (B ergeys
manual, Kauffmann, Edwards-Ewing) have certain
differences, the general approach is the same. The
c.lc]ilLv is first classified into its mayor subdivision -
C o p y rig h te d m aterial
group nr trihe. Each Irihc consists g f one or m art feiglttnnb, F. hcrmxnii and F . i-y/mens which have
genem and each genus one or more subgenera and been isolated infrequently from clinical specimens.
species. "I lie species arc classified iiuo types— E. blittAC found in rhe gut of cockroaches is
bin types, serotypes, bacteriophage types, colicin biochemically different in being indole and beta-
types, galactoridasc negative, lr has not been isolated from
clinical specimens.
DNiFE :T£S: MffTS E Unlike other colifomis, £ coins a parasite living
Tribe h Esch^richiae only in the human Of animal intestine. Voided in
Genus 1. Escherichia feces, it remains viable in fhc environment only for
2, EdwardBiella some days, Detection of E. cob in drinldng water,
!>. C iembaerer therefore, is taken as evidence of recent pollution
4. Salmonella with bumun or animal (tecs,
5. Shigella E . coJj is a Gram negative,
Tribe H: KJcfcidlcac straight, rod measuring 1-3 * 0 .4 -0 .7 pm arranged
Genus 1. Klebsiella singly or in pairs. It is motile by pernrichaTe flagella,
2 . Eoterobacter though some slums may he non mot Lie. Capsules
3. Hafhia and hmbrine arc found in some strains- Spores arc
4. Serratia not formed.
Tribe HI : Pfocecac It is jtl aerobe and
Genus 1- Proteus a facultative anaerobe. The temperature: range is
2- Morganclla 10-40 aC (optimum 37 aC), Good growth occurs
3. Provide ncia on ordinary media. Colonics ate large, thick, greyish
Tribe IV: Erwinieae white, moist, smooth opaque or partially iranriucent
Genus 1. Erwinia discs. This description applies to the sin(jCrth (SI
The genus Verrini'j, including [he plague form seen on fresh Isolation, which is easily
bacillus, has been placed in ihe family cmidriff ihY in saline. 'Ilie rough ■!K) farms give
E n tcrobaclcriaccac but because of the special rise to colonics with an irregular dull surface and
importance of plague, the major disease caused hy arc often autoaggln finable in saline. 1’hc 5 -R
yerunue and ire lack of similarity to enteric disease, variation occurs as a result of repeated subcultures
it is dealt with separately. and is associated with the loss of surface antigens
anti usually of virulence. Many pathogenic isolates
ISGHISISHM I'.T-U have polysaccharide capsules, Some strains may
This genus is named after Escherich who wm the occur in the "mucoid1 form.
first to describe the colon bacillus under the name Many strains, especially those isolated from
Bicterium cob commune ( 1 5). Based on minor pathologic conditions, are hemolytic on blwd agar.
differences m biochemical characteristics, colon On MacConkey^a medium, colonies art bright pint
bacilli were- described under various names but in due to lactase fermentation. Growth is largely
view of the ttt.j t i b i l i t y of the bii ichcm ical properri e* inhibited on selective media such as IJCA nr SS
in thi& group, they have all been included in one agar used for the isolation o f aalmonellae and
species E sch erich ia luJj which is further sliigellae. In broth, growth occurs as general
subdivided into biutypes and serotypes. A few other turbidity snd n heavy deposit, which disperses
species have been described In the genus bur they completely on shaking.
ire of little medical importance. These include F. G lucose, lactose.

C o p y rig h te d m aterial
nunnito], maltose and many other sugars arc
fermented with the production rtf sioid Jnd gst$.
Typical strains do nor ferment sucrose. The tour
biochem ical rests widely em ployed in [lie
classification o f entero bacteria are the indole,
methyl red (M R )t Vbget-Proskauer (V P ) and
citrate utilisation tests, generally referred to by the
mnemonic 'IM ViC', E, cob in indole and M R
positive, and VP and citrate negative (IM ViC + 4
— ). Gelatin is not Liquified, is not formed,
urea is not split and growth does not occur in KCN
medium.
Serotypirig of E, coU is
based on three antigens - the somatic antigen O,
the capsular antigen K and the flagellar antigen H.
So far some 170 types o fO antigens, 100 K antigens;
and 75 II antigens have been recognised, Tile
antigenic pattern of a strain is recorded as the
number of the particular antigen it carries, as for
example 0111: K5S: H 2.
The K antigen is the acidic polysaccharide
antigen located in the 'envelope' or TnicrtHiapsulr.
(K tor Kuptcl, German for capsule). It encloses the
O antigen and renders the strain inagglutirtable by
the O antiserum. It may also contribute to virulence
by inhibiting phagocytosis. Formerly K antigens
were subdivided into three kinds - the thermobbile
L antigens, the thermostable A antigens and the B
antigens found on em ero p ith o g en ic strains
associated with infantile diarrhea. Later it was
shown that the B antigen was not a separate entity.
K inti gens sure therefore currently classified into
two groups, 1 and 11, general Iv corresponding to
rhe former A and 1. antigens.
Several different serotypes of E. cob are found
in the normal intestine. Most of them do not have
K antigens. The normal colon strains belong to the
'early' O groups (1 , 2, 5, 4 e tc .), while the
enrernpithogtmc strains Lielong I d the 'Later' o
groups (26, 55, 86* 111, etc)
Two type? o f virulence
factors have been recognised in E- coli— surface
antigens and toxinsc
The som atic lipopolysaocharidc surface Q
antigen, besides exerting endotoxk activity, iJso
protects die bacillus from phagocytosis and the
bactericidal effects o f complement. The envelope
or K antigens also afford protection against
phagocytosis and antibacterial factors in normal
■serum, though ;E is not effective in the presence of
antibody to O or K antigen Most strains of E. CQli
responsible for neonatal meningitis and septicemia
cany the Kl envelope antigen which is a virulence
factor resembling the group H antigen of
m eningococci.
Fimbriae also promote virulence. The common
fimbriae seen in most enterobacteria, which are
ehromosoinuliy determ ined, present in Large
numbers and causing mannose sensitive
Lren[[agglutination, are probably not relevant in
pathogenesis. A different fond o f iimbriae which
are plasmid ended, found only in small numbers
and mediating mannose resistant hemagglutinins
have been shown to act as virulence factors. Some
id them may not ticeuras morphologically separate
structures but only ss surface antigens -f o r example,
K88 and K99 antigens in strains causing diarrhea
in animals, or tbe Colon j'saricui Factor Aulfgifjjs'
(CFA) in enterotoxigenic E. coli causing human
diarrhea. Fimbriae arc also o f importance ill urinary
tract infection, as for example the P fimbria which
binds specifically to the F blood group substance
on human erythrocytes and uroepithelial cells,
£ , raff produce two kinds o f exotoxins -
hejnofysi'ju and enteruttudm. Hemolysins do not
appear to be relevant in pathoge rtesi h thougLi they
arc produced more commonly by virulent strains
than by 1 virulent strains. J’ nterotnxiiis .itc mipnrCLiH
in the pathogenesis of diarrhea. Three disrinct types
of Er cob entcrotosins have been identified - li« t
labile toxin (L T ), hear stable toxin (S T ) and
verutuxin (V T ) itso known as Shiga-like toxic]
(SLT).
The E. aoti cnierotoxin L l was discovered in
1^56 by De and colleagues in isolates from adult
disrrlbCa cases in Calcutta, by the rabbit ileal loop
C o p y rig h te d m aterial
 Motility + + * - - 4 t t t t t
das from glucose + + 4- - 4 t + d d t 4
Add from lactose 4 - 4 - - *■ t - - - - -
Add from sucrose d - d - - 4 * - 4 d - d
Growth in KCN - - + d - * * * * 4 + +
Indole + + d - d - - - - d 4- 4-
MR t + 1- f + - - - - 4- + f
VP - - - - - 4- 4 -4 4- - - -
Citrine - - 4- - * 4- 4- d d d
H IS - + 4- + - - - - 4- - -
Urease - - - - - * d - - * + d
PbenyUlinine
deaminase (PPA) - - - - - - - - - 4 + 4-
Arginine
d-chydmlase d - d + - - d - - - - -
Lysine
decarboxylase + 4- - + - J d * 4 - - -
Ornithine
decarboxylase d t d t d —■ * ■4 t d *

[d - results different in different species nr strains.}

KnptHtit:
JS. (yphi does not produce gat from ngsn,
’Sh. sonnei ferments EactoBe and sucrose late
method which they had earlier used for identifying
the cholera, enfemtoxin (CT), vi-z. injection of E.
cofj culture filtrates into dosed ligated loops of rabbit
ileum induced outpouring of fluid and ballooning
o f the loops, E, t oh LT resembles- the cholera toxin
in its structure, antigenic properties and mode of
action. It is a complex of polypeptide subunits -
each, unit of the t o m consisting of one subunit A
(A for arrive) and five subunits B (B for binding).
Tile toxin bhids to the G m l gangliosidc receptor
on intestinal epithelial cells by means of subunit B,
following which the subunit A is activated to yield
two fragments - A l and A2. "Hie A l fragment
activates adenyl cyclase in titc enterocyte to form
cyclic adenosine 5' monophosphate (cA M P ),
leading to increased outflow o f w ater and
electrolytes into the gut lumen, with consequent
diarrhea. Though [lie mechanism of action o f LT
and C T is the &ame> the latter is about a hundred
times more potent than the former. LT is a powerful
antigen and can therefore, be detected by a number
of serological as well as bio-logical rests (Table
30.2).
Tbe best stable toxins of E- Cfl/f (5T \ first
identified in 19 7 0 . are low molecular weight
|K>lypep[ides which are puorly antigenic. Two types
of ST are known, STA (or ST 1) and STB (or ST
II). ST^ acts by activation of cyclic guanos!ne
monophosphate (cGMPJ in [he intestine. It acts
very rapidly and induces fluid accumulation in the

C o p y rig h te d m aterial
 i E ntp rab acten aceS G I: Colirorm s - ProlfluS
intestines o f infant mite within Four hours of
intragastrie administration. This mf.iril mouse test
is the standard method for demonstration of ST ,.
It also Induces fluid accumulation in the intestinal
loops o f neonatal hut ro t weaned piglets- S T , is
methanol soluble. S T Hcauses fluid accumulation
in young piglets (uptn nine weeks) but not in infant
mice. The mode of action of ST n is not known but
it is nor through cA M P or cG M P. It is not
methanol soluble. ST genes, ait carried on plasbiuLs
which may also cany other genes, such .1- for I T
and drug res stance. However, ST. and STa genes
are not seen to be carried on the same plasmid.
E, colt Vcrocyiotoxin or Veroroxin (V T ) was
so nairted bec&LLSe il v\,l> dlj■-Ldetected (1977) by its.
cytotoxic effect on Veru cells, a cell line derned
fmm African green monkey kidney cells. It is also
known as Shiga-like toxin (SLT) because it is
similar 10 the Shif^elln dyvenKiixe type L roiun in
its physical, antigenic and biological properties.
Besides cytotoxicity in Vero or H eLa cells, VT also
shows enterotmricity in rabbit ileal loop- and mouse
paralytic-lethality as does the Shiga toxin. V T is
also composed of A and B subunits. V T genes
appear to be phage encoded. An antigenically
different VT, called V T . Kue been idem 1lied, which
is not neutralised bv the Shiga antitoxin, unlike
VTr
C l in ic a i I n fe c t io n s
Font main types of clinical syndromes are caused
by E. cnli: 1) urinary tract infection, 2) diarrhea,
3) pyogenic infections, and 4) septicemia.
U r i n a r y t r a c t in fe ctio n ! E, cob" and other
eollforms account for the large majority of naturally
acquired urinary tract infections. Those acquired
in the hospital, following instrumentation, ire more
often caused by other bacteria such as /'scudomonas
and PtvTeui.
The E. coir serotypes commonly responsible For
uTinary tr.n i infections are those normally found in
the feces, O groups I, 2, 4, G, 7, etc. Only one
serotype is generally isolated from infected urine at
t 275
a time* though recurrences may be due to different
serotypes.
Irifecrion may be precipitated by urinary
obstruction due to prostaric enlarge^umf, calculi
or pregnancy. About 5 - 7 per cent o f pregnant
women have been reported to haw urinary infect ion
without any sym ptoms. Such asvm prom airf
bacfcmiria undetected and untreated may lead to
sym ptom atic infection Later in pregnancy,
pyelonephritis and hypertension in the pregnant
woman, as well as to prematurity and perinatal
death of the fetus.
While infections of the lower urinary' tract seem
to be 'ascending infection caused by fecal eollforms,
pyelonephritis is probably due to hematogenous
infection. Strains earning K antigens are more
commonly rcspon-'ible for pyelonephritis while
most isolates from cystitis lack K. antigens.
B acteriological diagnosia of urinary tract
infection has undergone a marked change following
the developm ent by Kass o f the concept of
'significant bacteriuri.L1. Normal urine is sterile hut
during voiding may become contaminated with
genital com m ensals. In order to avoid such
contam ination, urine used to be collected by
catheterisation for culture. Any bacterial growth
from catheter!'icd urine was considered to denote
infecrion- Even under ideal cond itions,
catheterisation leads to urinary infection in at least
two percent, and when precautions are inadequate,
the risk is much higher. Hence catheterisation Is
no longer considered justifiable for diagnostic
purposes. Instead, dean-voided midstream samples
H>f urine are employed for culture. Siii I: xpv. liOciIk
should be collected carefully to reduce
contamination to the minimum. In men, it is
sufficient if midstream urine i-s collected after the
prepuce is retracted and the glans penis deaned
with wet cotton. Tn women, anogenital toilet is more
important and should consist of careful cleaning
with soap and water Nemirriiant antiseptics such
as chlorhexidine have been recommended for vulval
cleaning. Unite should be passed keeping the labia
C o p y rig h te d m aterial
376 * Textbook of Microbiology
Table M .2 Methods lor detection ol ETEC
Assiy
frt mV? rests
Ligated ra b b iT ileal loop
Read at & hours
Read at 18 hours
Infant rahbir bowel
Infant mouse Lntragasnu: {4 hours}
Adult rabbit skin (vascular permeability factor)
In ^Htno tests
Tissue c u lt u r e rests
Rounding of Yl mouse adrenal cells
Elongation of Chinese hamster ovaiy (CHO) cells
Serological tests
■ELISA
Pissh»e agglutinati'in tests, passive immune
hemolysis, precipitin (Eileen's) test
Generic tests
ONA prunes
s-L-p^ir.L^L^cJ bv tinmen;. The first ptirtiim of urine that
flushes out commensal bacteria from the anterior
urethra is discarded. The next portion of the urine
(midstream sample] is Collected directly inct 1
Sterile wide mouthed container and transported to
the laboratory without delay. Urine is a good
medium for the growth o f coliforms and other
urinary pathogens, and hence delay in processing
will vitiate the results of quantitative Culture. If
delay of more than 1 -2 hours is unavoidable, the
spcci linen should be refri^eruted-
In quantitative cultures, m idstream utme
samples will give a hiphasic distribution ofcoJomes,
most specimens containing either less than 10,000
or more than 100,000 bacteria per ml. Kass and
other investigators have established that in the
presence of active infection in the urinary tract the
urine will contain 100,004 bacteria or more per
ml. This level is, therefore, considered to represent
aijgttificarif iMcfenun'i Counts of 10,000 bacteria
or Cess per ml are due to contamination dining
ffrimfcndni
LT ST
* 4
4 -
4 4
- 4
+ -
-
4 -
41 (ST-EiiSA
with monu-
colonial
-
antibody
-
4’
4
4
voiding and are of no significance. Counts between
the two levels are infrequent when the sample is
collected properly and processed promptly. Such
results should he considered equivocal and the
culture repeated. Needless to say, interpretation of
tjacteriuria, should always be with reference to the
condition of the patient. In patients on antibacterial
ordiuTeric drugs and with some bacteria like Staph,
aureus, even low counts may be significant-
Rsr quantitative culture, serial ten fold dilutions
of urine are tested hv the pour plate or surface
culture methods. Thj*, however, is too complicated
for routine diagnostic work, for which
senaiquandraiive techniques ate more convenient.
The most widely used techn ique employs a standard
loop which transfer* a fixed, small volume of urine.
One loopfld of urine is placed on a lion inhibitory
medium (blood agar] and another loopful on an
indicator medium (M acConkey). The former
medium gives a quantitative measurement o f
hacterinriar while the latter enables a presumptive
C o p y rig h te d m aterial
 * Emenobactenac&ae h: CotiEorms - Proteus ►
di.igno^i^ o f the bacterium . T h e [solutes ire
identified by their properties.
Bacteriological investigation o f urinary tract
infection is not complete without an antibiotic
sensitivity test of the isolate. E. coti and other
common urinary pathogens develop drug resistance
so frequently that no anfihacterijl therapy can be
instituted meaningfully without testing individual
strains. Resistance is often to multiple drugs and is
o f the transferable variety. Antibiotic sensitivity tests
may be done directly using the uri:ie samples as
inocuta and the results confirmed by repeating the
test with individual isolates.
Because urinary Luc Li rdL-ct ion is such a common
problem and bacteriological facilities are not always
available, several screening techniques have beet)
introduced for the presumptive diagnosis o f
significant hacteriur'n-t. These include the following:
1) Gricss nitrite test—based on the ahsence o f nitrite
in normal urine. The presence o f nitrite, detectable
by a simple test, indicates the presence o f mitiite-
rcducing bacteria in urine, 2) catalase test — the
presence o f catalase as evidenced by frothing on
addition of hydrogen peroxide indicates bactcriuria,
though a positive result is obtained also in
hematuria, 3) triphcnyfrcrraxoiiumchloride; [ T O
test - based on the production o f a pink-red
precipitate in the reagent caused by the respiratory
activity o f grow ing bacteria* 4) microscopic
demonstration o f bacteria iu Gram stained films o f
urine, 5) glucose test paper - based on the utilisation
o f the minute amounts ol glucose present m normal
urine, by bacteria causing the infection and 6) dip
slide culture methods - agar coated slides arc
immersed in urine or even eicposed to the stream of
□tine during voiding, incubated and the growth
estimated by colony counting or by colour change
of indicators. None o f the screening methods ia as
sensitive or reliable as a culture.
T h e antibody coated bacteria test has been
employed for the localisation o f the nice a f urinary
infection. This is based on the assumption that
bacteria coated with specific antibodies are present
277
in the urine only when the kidneys are infected
and not when the infection i* confined to the
bladder. Antibody railed bacteria are detected by
immunofluorescence lining fluorescent tagged
antihum an globulin or by staphylococcal
coaggliitmaTiirn.
D ia r r h e a : Right from 1885 when Eschcrich
fust isolated the bacillus from the feces o f infants
with entrriri^, E, coii had been suspected to be a
causative agent o f diarrhea. However, as there was
no way then o f differentiating diarrheagenic E . coli
strains from the welter o f commensal strums
invariably present in normal feces, it remained
unconfirmed till serotyping schemes for E. coli
were developed. It was only in 1945 that Bray
established the etiological role o f a specific type of
ELcoJ^subsequently recognised as type 0111) during
a hospital outbreak o f childhood diarrhea m
London. Soon many other enceropathogemc
serotypes o f E, coti came to be recognised as
responsible for infantile diarrhea. Subsequently,
other varieties o f E. coli diarrhea came to be
identified in children as well as in adults. A t least
five different types, of di mrheagei llc E, cob .ire now
recognised - entcropathogemu', enterotoxigenic,
enteroinvasive, entero-hem orrhag ic
(fihigatoxigeiiic or Verotoxigenic) and entero-
aggregative E, coti.
h i t t e r o p u t h o ^ n i c E . c n li ( E P E G ) : T h e se
have been associated 11 ll:.i nly wl1h diu rrhe ,lin i flfants
and children usuallv occurring as institutional
outbreaks but they can also cause sporadic diarrhea
in children and lets often in adults, E P E C diarrhea
was co nun on worldwide from the late 1940s to the
19b0s. Afterwards, it has become less common,
E P E C were identified by serotyping. initially
by their O and B antigens (for example, 02t:B 6h
055:B5 f 0111 :B4 and so on). After the existence of
the B antigens became suspect, only O typing
practised.
The diagnosis o f E P E C diarrhea is relatively
easy during outbreaks but very difficult in sporadic
eases. Fresh durrheal feces is plated on blood agar
Copyrighted material
27$ * Texmook o< Microbiology
and M a c C o n k ^ r media. A lter ■ iw m i^ k l inaibado n,
E . coli colonics arc em ulsified in mi line on a slide
and tested fo r agg lutinatio n by p ^ l\ t ;l Il-nt and
monovalent E P E C O antisera. A t least ten colonics
per plate should be tested as many serotypes are
present in a single culture. I f isolated colonies are
negative, the confluent growth is cm u l^fied and
tested. D u rin g outbreaks, if tbe causative serotype
ls know n, cultures need be tested only w it h the
p articu la r anti>etu m . E P E C an tise ra are now
d iffic u lt to o b tain and so specific d iag n o sis is
av ailab le o n ly in few laborato ries. W h e n the
outbreak is caused by a strain w ilh some readily
dem onstrable feature such as failure to ferment
sorbitol, rapid identification is possible by using
appropriate culture media.
T h e patho^ene^s o f E P E C diairhca is not fully
un d e rsto o d , E P E C do not o rd in a rily produce
e n te ro to \in sn nor arc they invasive- In infantile
enteritis, the bacilli are seen ro be adherent ro the
mucosa o f the upper sm all Intestine, intim ately
attached to cu p -lik e projections ('pedestals'! o1 the
entemeyte m em brane, causing di-^roprion n f the
brush border m icrovilli. T h e name enre n W h cre n f
E , co Jj' has been proposed for these scrams, w hich
can be identified by their adhesion ro H E p - 2 cells.
K n ie r o io 't i^ e n ic E. c o li (E T E C ):
D ia r r h e a caused by £ T E C is e n d e m ic in the
developing countries in the tropics, am ong all age
groups in the local papulation. Its seventy varies
fro m m ild w ate ry d ia rrh e a to fatal d ise a se
in d is tin g u is h a b le from ch o le ra. Persons from
developed countries visiting endem ic areas often
suffer from E T E C diarrhea - a condition known
as ‘traveller's diarrhea'. E T E C d:.urhea came into
prominence from the late l % t i s .
T h o u g h plasmklis w ith enteroto\in genes may
be present in any strain o f E . call, in praccice only
a s m a ll num ber of se ro ty p e s b e co m e
enterotoxigenic (for example, 0 6 ,0 6 ,0 1 5 ,0 2 5 ,0 2 7 ,
0 1 6 7 ) . T o x in production alone mav not lead to
illn e ss.T h e strains should fin t be able to adhere to
intestinal mucosa. T h is adhesion is mediated by
*
titiihn.il or colonisation factor amiigens, o f w hich a
number have been i den Tilled ( C F A I, I I , I T I J V ) .
D iagnosis o f E l E C diarrhea depends on the
demonstration o f entcrotovin in E . oof! isolates by
any o f the methods luted in Table 30.2. A strain o f
E T E C m ay produce either L T or S T or both.
Detecrinn o f L T is easy as many in vu:ro methods
are available, such as Tissue culture tests (rounding
o f Y . mouse adrenal cells and elonganon o f C H O
c e lls d u e to in tr a c e llu la r in cre a se o f r A M P
co n ce n tra tio n ), and sero log ical tc^ts ( K U S A ,
passive agglutination and im m unolysin tests). In
v ito tests such as rabbit loop or in trader io . lI tests
may be used when in vitro tests are not available.
T h e detection o f S T is more difficult. T h e infant
m ouse test is stilt w id e ly em plo yed. T h e poor
antigenicity o f S T has prevented the development
■ uf sero lo g ica l rests, [hough S I E L IS A using
monoclonal antibody has been introduced. Generic
probes are available for detection o f S T and L T in
Ei c cl; cultures, or directly in feces, food or water.
E n t e m i n v i f i v e E . c o li ( E l E C ) : T h e se
resemble shigellae in m any respects. M a n y o f these
strains are nonm otile, do not ferment lactose or
ferment it late w ith acid, but w ithout producing
any gas and do not form lysine decarboxylase. M a n y
o f these show O an tig en cross reactio n w ith
shigeliae. T h e se 'atypical’ E, coli strains had earlier
been giouped under the 'A lkalescens-D ispar G ro u p 1
and gLven nam es such as ‘SbigeNa a/JtaJescens
(re se m b lin g Sh. H cxncri except in fe rm e n tin g
dulcitol and form ing alkali in litm us lm lk) and ‘S h.
etapar' (late lactose fermenter like Sh, sonnei but
indole positive). Besides these 'atypical strains1 many
typical E. culi strains can also cause clinical illness
resem bling shigellosis. T h e se have been termed
enfenor.nia.sjir E . coli because they have the capacity
to invade interstitial epithelial cells in vivo and
penetrate H e L a cells in tissue culture, E I E C strains
usually belong to semigroups 02 B ac, 0 1 1 2 ac, 0 12 4 ,
0 13 6 , 0 14 3 . 0 1 1 4 , 0 1 5 2 , 0 15 4 .
Clinically EIEC infection resembles shigellosis,
ranging from mild diarrhea to frank dysentery, and
Copyrighted material
occurs, in children well a-. adults. F ot laboratory
diagnosis o f F I E C , the Sereny test u-secl to be
em ployed (that is, instillation o f a suspension o f
Freshly isolated E I F C or shigella into the eyes o f
guinea pigs leads ro mucopurulent conjunctivitis
LinJ severe keratitis), M ice may be used instead o f
guinea pigs-. C e ll penetration <>t H e l.a or H E P - 2
cells in culture is a more humane diagnostic test,
T h is ability to penetrate cel In is determined hv a
large plasm id, detection o f w hich can also be a
d ia g n o s tic test- T h e p la sm id codes for o u te r
m em brane antigens called the 'ninrlenct m urker
antigens* ( V M A ) w hich can. he detected hv the
E U S A ( V M A E L I S A ) teat.
E , m li. an*
E .c o li
strains producing verocyiOMMtiii ( V T ) Of S li iga- Like
toxin ( S I T } can give rise to durrhc.il disease ranging
in severity from m ild diarrhea to fatal hemorrhagic
colitis and hemorrhagic uremic syndrome ( H U S )
particularly in young children and the elderly. T h e
prim ary target for V T appears to he the vascular
endothelial cells-T h is m^y explain rhe pathogenesis,
o f H U S , in w hich a characteristic renal lesion is
capillary m icroangiopaflnc V T E C also produces
diarrhea m eartle and pigs. T h e typical E H E C is
serotype 0 1 5 7 : H 7 , hut a few others such as 0 2 fr:
H i also belong to this category.
T h e disease m ay o ccu r s p o ra d ic a lly or as
o u tb re ak s o f fo o d p o isn n o n g . T h e source of
infection is contam ination by hum an or anim al
feces, directly or indirectly. C h an g in g life styles and
eating habits, wish growing p o p u larly o f fast f w t L
have led to a remarkable increase in E H E C (bod
poisomng. 1 n the U 5 A , about 20,000 cases o f 0 :1 5 7
ft]od p o is o n in g o c c u r every vear, m an y w ith
hem orrhagic com plications. A large outbreak o f
food poisoning caused bv E . co /j 0 : 1 5 7 , with OVW
10 ,0 0 0 cases occurred in Japan in mostly
affecting school ch ild ren - In vestig atio n of this
epidemic revealed the source o f infection to he salad
vegetables such as radish and alfalfa sprouts, in
w hich the bacteria were found beneath the skin
and in the deeper tissues. W a sh in g w ould not
rem ove the bacteria from such vegetables and
co o king alone m ay ensure safety. T h is fin d in g
extends rhe scope o f E H E C food poisoning to
Vegetarians also.
Laboratory diagnosis o f V T E C [liar rhea can be
made by demonstration o f rhe hacilli or V T in feces
directly or in culture. A s V T E C form s o n ly a
m inority o f fecal coliforms in infected cases, testing
in d iv id u a l colonies may not he successful. T h e
sensitivity can be considerably increased by using
D N A probes for V T j and V T . genes. V T can be
delected bv its cytotoxic effects on Vero or H e L .i
cells. Dem onstration o f V T neutralising antibodies
in co nvalescent sera m ay help in retrospective
diagnosis.
M o st V T E C strains belong to the serotype
0 1 5 7 :H 7 w hich does not ferment sorbitol, unlike
the m ajo rity ot E . call. So the use o f sorbitol
M a cC o n k e y m edium helps in screening for 0 :1 5 7
VTEC.
T h e se strain* ;ltv so nam ed because they appear
aggregated m a 'stacked brick’ formation on H e p -
2 cell* or glass. T h e y have been associated w ith
p e rsiste n t d ia rrh e a , e s p e c ia lly in d e v e lo p in g
countries. M o st o f the are O -uncypable, but many
are H -tvp ab le. In anim al experiments they cause
shortening o f villi, hemorrhagic necrosis and mild
edem a w ith m o no nuclear in filtra tio n n f the
submucosa. T h e y form a low molecular weight heat
■ stable e n t r r o to x in c a lle d EASTl
{ L-eiitcroagm«g3itT,vE licat srabfe c n flcn stu jd n -l}.
E . coli form the m ost
com m on cause o f intra-abdom inal infections, such
as peritonitis and abscesses resulting from spillage
o f bow el co n te n ts. T h e y also cause p y o g e n ic
in fe c tlo n s in the p e ria n a l area. T h e y are an
important cause nfnennatal meningitis.
B lo o d stream invasion by £ r p d i
may lead to fatal conditions like septic shock and
W tcm ic inflammatory response syndrome ESJRSh
Copyrighted material
 A r £ r cnii com m only show m ultiple drug
resistance, antibiotic sensitivity testing of strains is
important in ire accident.
antigen serological I v identical to th e antigen o f S.
used for
Jyphi and 5 , paratyphi C - T h e se m sy he
the estimation o f V i antibodies or for raising V i
antisera.

The genus contains the species EdwardsicUa
&rda which is a noncapsulated, motile bacillus w ith
weak fermentative powers. The name farda refers
to its tardy or weak fermentation o f sugars. O f the
si.]gars cum raorilv used, only glucose acid maltose
arc fermented. It forms indole and H ^ S , utilises
citrate and decarbaxylates lysine and ornithine.
E . tarda is a normal intestinal inhabitant of
snakes and other cold blooded animals. It has been
cultured from normal and diarrhealc human feoes.
Its pathogenic role Is uncertain but it has been
isolated from wounds, urine, blood, and from CSF
in cases of fatal meningitis.
These are motile bacilli which utilise citrate, grow
in KCN m edium, produce H 2S and ferment lactose
late or not at all. Three species are recognised, C i t m
frru n d ii which gives typical reactions and Citio,
ioseW (formerly Cj'rro. diverses) and Cifro.
tmiJonitrois which d o not fo rm H 2S- C j> ™ .
fre u n d ii strains were form erly classified as the
'Ballemp-Bethesdi group1. They exhibit extensive
antigenic sharing with salmoncUac. T h is may cause
Cirrobacter is a normal intestinal inhabitant.
Many strains formerly called paracolons belong to
this group. It has been isolated from a few cases of
enteric fever but its etiological role is not established.
It may cau&e infections o f the urinary m et, gall
bladder, middle ear and meningCS-
f. i M i e i J L f : .
T h e genus Klebsiella consists o f non m otile,
capsulated rods that grow well on ordinary media
forming large, dome shaped, mucoid colonics of
varying degrees of stickiness. They are short, plump,
straight rods, about 1-2 k 0.5-O.fl mm in size. The
capsule is often prominent and can be made out
even in G rim stained, smears as haloes around the
bacilli. Klchsiellae are widely distributed in nature,
occurring both as commensals in the intestines and
as saprophytes in soil and water. T h e ir classification
has undergone various modifications. They have
been classified into three species based on
biochemical reactions and into twet 80 serotypes
based on the capsular (K) antigens (Table 30.d).
lbL.15BSj| EiUL S, M TONI !£ £

confusion in the diagnostic laboratory. Some strains [bhi-dlander's- baolltu , Bacillus mucosus capfuiattis)
(for otmipte, the Bhamagar strain) have a Vi This bacillus was first isolated by Friedlander

Gas from glucose + d _

Acid from lactose + d -
MR - 4- 4-

VP + - -

Citrate + d -

Urease +■ d -
M donate 4 - 4-

Lysine 4- d -

C o p y rig h te d m aterial
{IriS .l) from fatal cases o f pneum onia. It ferment?
sugars {glucose, lactose, sucrose, m annitol) with
the production o f acid and abundant gas. It lS mdole
and M R negative and V P and citrate positive ( 1M
V i e -------* +)■ B io ch e m ica lly variant strains arc
com m on, It io n ns urease. Strains, formerly labelled
as non motile Aerobactvr aenogencs(K. aenogrjresX
are ro w considered to be K. pneum oniae subspecies
acrogencs, It is the second most populous member
o f the aerobic bacterial flora o f the hum an intcttinc-
I t has b e co m e a v e ry im p o rta n t cause o f
nosocom ial infections, even replacing E . lujj in
some centres. Jr causes pneumonia, urinary'infection,
other pyogenic infections, septicem ia and rarely
diarrhea-
KlcbsicH? pneum onia is a serious disease w ith
high case fatality, It occurs in m iddle aged or older
perso ns w ho have m edical p ro blem s su ch as
alcoholism , chronic bronchopulm onary disease or
diabetes m eilitus. T h e disease is characterised by
massive mucoid inflam m atory exudate o f Lobar or
lobular distribution, involving one or more lobes
o f the lung. N ecro sis and abscess form ation arc
more frequent than in pneumococcal pneumonia.
Serotypes 1 , 2 and 3 are usually responsible for
pneum onia. Positive blood cultures can be obtained
in about 25 per cent o f the cases.
K. pneum oniae Is a frequent cause o f urinary
infection. A s most strains art resistant to antibiotics,
treatm ent poses serious problems. It also causes
pyogenic infection?, such as abscesses, m eningitis
and septicemia.
Som e strain? o f K , pneum oniae isolated from
cases o f diarrhea have been shown to produce an
cnterotoxin very sim ilar to the heat stable toxin o f
G as from glycerol - +
Acsculin hydrolysis - 4
] . y s i n e d c c ^ f b c h x .y L a s e “
Arginine dihydiuLlK 4-
E , call. T h e production o f this toxin is determined
hv the presence o f a plasmid.
D ia g n o sis i? m ade by cu ltu rin g appropriate
s p e c im e n s and id e n t if y in g the is o la te by
biochem ical reactions. A n tib io tic sensitivity should
invariably be done, M a n y strains ca n y plasm ids
determining m ultiple drug resistance.
K. ozaenae is a bacillus associated w ith ozena,
a disease ch aracte rise d by foul sm e llin g nasal
discharge. Identification is difficult due 10 wide
variations in the biochem ical reactions o f individual
strains, IC ozaem e belongs to capsular types 3 -6 ,
K . rhjjiosclero-m afjs causes rhinosclerom a, a
chro nic granulom atous hypertrophy o f the nose
prevalent in southeastern Europe, In d ia and Central
A m e rica . T h e b acilli are seen im ra ce llu la rly in
lesions. It car be identified by biochemical reactions
and helongs to capsular type 3.
T h e species A'. axytoca m ay be rarely isolated
from clinical specimens.
Form erly known as Acrobacter, these ate m otile,
eapsulatcd, Lactose Fer men ring bacilli w hich are
indole and M R negative and V P and citrate positive.
Tw o clin ically relevant species are E. d M f i f and
F. . aert^enes (Table 30-4).
They are normally found in feces, sewage, soil
and w ater and rarely in urine, pus and oth er
pathological materials. They may be responsible
fur hospital infections.
T h is is a m otile, n o n lacto ^c-feiuienting baciUu?
which is indole and M R negative and V P and citrate
4
C o p y rig h te d m aterial
positive, Uiochcmica] reactions are evident bent
at 22 CC; at 57 1C diey may he d e r iv e or irregular.
O nly one genus is recognised, H, a t m It is
found in human and animal feces, sewage, soil and
water.
b £ ", |&
T h b differs from Hafaia in forming a pink, rtd or
magenta, nmdiflusible pigment called prodigies in
which in formed optimallv at room temperature.
O nly one species is of medical importance -
m arccsccns ["Bacillus p r o d i g i o s u s Jt is
pleom orphic, w ith m inute eoceobacillfliy and
normal haeilkair forms. I t b a saprophyte found io
water, soil and fined. Ir may grow in sputum after
coUcctiou and may suggest hemoptysis because of
the pigm ent form ed ('pseudohem optyfils').
Nosocomial infections due to Sr martest^™ are
heirg reported w ith increasing frequency. The
bacillus has been associated w ith meningitis,
endocarditis, septicemia, peri ton it in, respiratory'
infection and many other conditions. Muluple drug
resistance is common in hospital strains.
7 -i 3-3f1vo I'IL' 3
(Proteus bacilli)
Proteus bacilli constituting the tribe- Pmtccac are
lactose nonfermenters and so do not strictly belong
ro rhtr group o f ‘col i form bacilli", However, they
are also norm al intestinal commensals and
opportunistic pathogens like coliforms, and so are
included in this chapter. The name 'Proteus1refers
Urease +■
Ornithine +■ - ■+
decifboxylue
Indole - +
Fetmentanon of d o d io l - -
Fermentation o f trehalose + t
to their pIcomorphiniTi, after the C re e k god Pfoteus
who could assume any shape.
T h e tribe Pro frcaeis classified into dim e genera
- ■ Prureus, jllorg-jneJ/a and fVovrd'eiicaa. M o st o f
them except for fiotoe P runndenciu strains, produce
a powerful urease w hich rapidly hydrolyses; urea
10 am m onia and carbon dioxide. A characteristic
feature w h ich distinguishes Ptote&ae from o ilier
enterobacteria is the presence, in all members of
the tribe, of the cnavme phenyl alanine deaminase
w hich converts phenyl alanine to phenyl pym vic
a cid ( P P A re a c tio n ). A l l o f them w ith few
exceptions show the following features:
G ra n t negative, noncapsulated, pleom orphic,
motile itjds,
* resistant to K C N :
■ degrade tyrosine;
* fail to acidify lactose, dulcitol or malonate;
' do CLUl form arginine Of lysine decarboxylase or
b ™ galactosidasc;
* M R positive, V P negative.
T h e mu-jot differential ing features o f m edically
important species o f proteus hacidi arc shown in
T ab ic 30 .5 .
Proteus bacilli possess somatic O and flagellar
1 1 antigens, w hich are o f considerable historical
interest. W e il and Felix ( 1 ^ 1 6 ) studying Proteus
bacilli observed that flagellated strains growing on
agar formed a thin surface film resem bling the m ist
produced by breathing on glass and nam ed this
variety the 1JJau ch r form (from llauck, m eaning
film of breath). Nonflageilated variants grew as
* m +■
- - -
■* * + t
- ±
i - + -
C o p y rig h te d m aterial
isolated colonies without the surface film and were
called 'O h n e H a u e b 1 (m eaning w ithout film o f
breath). T h e se names came ro he abbreviated as
the H and 0 forms. Subsequently the I i and O
were extended to refer to the flagellar and somatic
antigens o f other bacilli as well. "1 he terms are also
used To designate the type o f bacterial agglutination,
the H type referring to the loose, fluffy masses
formed when flagellated cells aie aggforinatet], anti
the 0 type to the fine gran u lar appearance o f
somatic agglutination.
W e il an d belt* also o b served that ce rtain
non m otile strains o f Pr, w lg u rh , called the LX
"trains', were agglutinated by sera from typhus fover
patients.T h is hetefrphilic agglutination doe to the
sharing o f a carbohydrate hapten by certain strains
o f Proteus and riekettsiau forms the basis o f the
W c il -F e lix reaction for the diagnosis o f som e
rickettsial infections. T h re e n o nm o tile Proteus
strains 0 X 2 , O X IV and O X K are used in the
agglutination test.
Proteus bacilli are w idely distributed in nature
as sap™ jphytes, being fountl i n dccumposi ng a ni rna I
matter, in sewage, in manured soil and in human
and animal feces. T h e y are frequently present on
the moist areas o f the skin. IT e y arc opportunistic
pathogens , com m only responsible for urinary,' and
septic infections, often nosocomial.
T h e genus Pboreujt co n tain s two species o f
m edical im portance - Pr. nrirabitis, w hich is an
important urinary ami nosocomial pathogen, and
Pr, vu/g^rfs w hich is found m uch less com m only
in hum an infections. F t. ftifpabj'Jfs in indole negative
iifid Pi, indole j>usiiivc.
C u ltures o f [iroteus bacilli have a characteristic
putrefuiclivc odour described asTish y' or 'seminal',
ft. mirabilis and Pr. vulgaris s m r m on solid culture
media. D iscrete colonics arc seen in young cultures
but thereafter actively m otile cells spread on tire
surface o f lire plate in successive waves to form a
thin b ln iv 1 :iyer in concentric circles. Sw arm ing
growth is a problem in the laboratory when mixed
grow th is obtained in w hich proteus b acilli are
present with other bacteria, Several methods have
been used to inhibit swarm ing - increased
co ncentration o f agar, in co rp o ratio n of ch lo ral
hydrate (1:5 0 0 ), sodium aaide (1:50 0 ) , alcohol ( 5 -
6 % ) , sulphonam idc, surface active agents or boric
a d d ( t 1 0 0 0 } S w a rm in g lin e s nnf o c c u r o n
M a c C n n key's m edium, o r w hich smooth colourless
colonies are formed.
The genun M ofganeU a has only one species,
M. m o igxn ii (formerly Pr, m oiga im ). it does not
swarm in culture. It is commonly found in human
and animal feces and causes urinary infection
infrequently Kosocomlal wound infections also
o ccu r
T h e genus P ro y id cn cia (form erly known as
Profcus rnconstens) contains three species seen in
clinical infections, ftw t ik a lih d en v is sometimes
seen in normal human feces but far mote frequently
in diarrheal Stools (hough its etiological role is
uncertain. Prov. slusrtii is ;l common cause oi urinary
infection and o f infection in burns. Prov- rettgerii is
part o f (he norm al fecal flo ra o f reptiles and
amphibians, and sometimes muses nosocomial
infection o f the urinary tract, wounds, bums and
blood.
Proteus bacilli arc resistant to many o f rhe
common antibiotic*. An exception is Pr. mirahilis
which is sensitive to ampkiUm arid cephalo-sporims.
ProviJenviaare the most resistant, particularly Prov.
which is also resistant to disinfectants such
s tu .irtii
as dilorhexiduu:, Oetrimide, benzalkonilim chloride
and heavy m crsl com pound^ such as silver
Eulphonamide, making it a major pathogen in hums
units, [i in sensitive io p fen v l and glutanildehyde-
Atnikacin and ciprofloxocin arc generally effective
in treatment.
These are anaerogenie bacilli forming a yellowish
pigment, usually found in sod and causing plaint
infections. E . iietb ico h has occasionally been
isolated from respiratory and urinary infections in
predispored or hospitalised patients.
C o p y rig h te d m aterial
C o p y rig h te d m aterial
 Ente robacte riaceae II: Shigella
Dysentery is a clinical condition o f multiple etiology,
characterised by the frequent passage o f blcK>d
stained, m ucopurulent stools. T h e two com m on
ty p e s o f dysentery arc bacillary and amebic. T h e
causative agents o f hacillary dysentcTy belong to
the genus Shigella, so named after Shiga, who in
18 9 6 isolated the first member o f this genus from
epidem ic dysentery in Japan. Som e other bacilli,
su ch as e nte ro in v a s iv e E- c o b , V ibrio
porahaemolytius and Campylobacter, can also cause
the clinical picture o f dysenterr.
S h ig eE lac are s h o rt, C r a i n
negative rods, about 0 .5 pm * 1 - 3 pm in size. T h e y
are n o n m o file , m m sporing and noncapsulated.
Fim briae may be present.
T h e y arc aerobe*
and facultative anaerobes, w ith a growth tempe­
rature range o f 1 0 -4 0 DC and optim a o f 37 ° C and
p H 7.4 . T h e y grow on ordinary m edia bur less
readily than other enterobacteria. After overnight
in cu b atio n , colonies are sm all, ahout 2 m m in
diameter, circular, convex, smooth and translucent.
O ccasio nally an primary' isolation and treqciendv
in subcultures, a proportion o f the colonics may he
o f the rough type. Co lo nies on M a cC o n k e y agar
arc c o lo u rle s s d u e to the absence o f lacto se
fermentation. A n exception is S / l sonnei which
ferments lactose I arc and forms pale pink colonies.
D e o x y ch o la te citrate agar ( O C A ] is a useful
selective m edium. Grow th is inhibited on W ilso n
and flair's bism uth sulphite m edium .
S h ig clk c are not specially resistant.
T h e y arc killed at 56 aC in one hour and by 1 %
phenol in 3 0 m inutes. In ice they last tor 1 - h
m onths.'("hey remain viable in moist environments
for days, but die rapidly on drying. In feces they die
w ithin a few hours due to die acidity produced by
the growth o f conforms. Sfr, sonnei is an general
more resistant than other shigella species.
S h ig e lla s are M R
positive and reduce nitrates to rti[rites. T h e y cannot
Utilise citrate as tile sole source o f carbon, do not
form I I S and are inhibited by K C N , Catalase is
produced, except by Sh. dyrrrtcerije Irpe 1. Glucose
is fermented with the production oi acid, without
gas, except lor the New castle and M an ch ester
biorypes of i ’h. ffevneri type 6, and some strains o f
S h . b o y Jii rvpes 1 3 and 1 4 . w h ich form gas.
Perm entation of m an n ito l in id im portance in
classification and diigell.ie have traditionally been
d iv id e d in t o m a n n it o l fe rm e n tin g an d
nonfermenring species. Sh, ilex non, Sh. boydii and
Sh, sonnet ferment m annitol, while Sh. dysertferiae
does not. Exceptions arc not infrequent. Lactose
and sucrose are not fermented, except bv Sh. sonnei
w hich ferments them late. A donitol, inositol and
fialicin are not lermented.
S h ig ctlae possess one
or more ‘m aior’ antigen) and a Large num ber o f
‘m inor somatic O antigens. Som e strains possess
K antigens. 'Fhese are not relevant in typing but
m ay sometimes interfere with agglutination by O
antisera. F im b ria ! antigens arc also present. In
general, the an tig en ic structure o f fih ig t U u is
sim p le , com pared to the com plex structure o f
salm o ndlae. There inconsiderable antigenic sharing
between Home m embers of the genus an well as
between shigelke and E . coii. C o m m o n fimbria]
C o p y rig h te d m aterial
antigeni may also occur, p u i k n l i i l y i n Sh. JTomeri-
l i in, therefore, im portant [hat the identification
of -^hiijeUac should he made hv a g o rn b iiU b cr of
antigenic and biochem ical properties and ro t by
slide agglutination ftloCK-
Shigclllae arc classified into tour species nr
subgroups based on a combination o f biochemical
find serological character]sties. Serotypes are
d istin g u ish e d w ith in The species. Sh. aonnei is
serologically homogeneous and is classified by
Cuticin typing.
T h is species
flf m annitol nontermenting bacilli consists of ten
sero type s, T y p e 1 is the b a c illu s o r ig in a lly
described by Shiga (S k . sht^ae}. Jr is indole negative
And is the only member o f the tamilv that ls alwavs
catalase negative. {Sh. tchmitzi and 'Sh, #o/uiei are
invariably catalase positive„ while am ong other
sh ig ella species, som e strain s may be catalase
negative,)
S h . d rsen tcrizc type 1 forms a toxin (Shiga
tnatm), the earliest eitai nple o f an c ra ta u n produced
by a G ra m negative bacillus. Three types o f toxic
activity have been demonstrated in shigella culture
filtra te s: ( 1 ) n e u ro to x ic ity , d e m o n stra b le by
paralysis and death l>h injection into m ice or
rahbifs. 'I‘hough known as WurntCfXm* the prim ary
site o f its action appears to be not the nervous
tissue hut the blood vessels, m ainlv o f the central
nervous system, with rhe neurological effects being
Secondary; (2) cmTerOIOXicitY, with induction o f flutd
accum ulation in Ligated rabbit ileal loop. Tw o
new shigella enterofoxins- have been id entified,
designated as S h . E T - 1 and 2, the form er
co nfined to S h. Qataeri 2a and the latter mote
widespread; and ( j ) cytotoxicity, c ausi ng cytopath ic
changes i m cultured Vitro Cells. T 1 Lis apjitani to be
the sam e as V erotoain 1 (or S h ig a -lik e toxin)
produced bv certain strains of £ . caii ( V T E C ). T h e
toxi n con sists o f binding (B 1 and active { A subun i ts.
Sub-unit A is divided into two fiig u icn ts A l and
A 2. fragm ent A l appears to inactivate host cell 60
S ribosome, interfering w ith protein synthesis.
S h . d y w n t c r i i c type 2 [ S h . t t h i t i i t z i ) form s
in d o le an d ferm ents so rb ito l a n d rh m n n o sc.
Serorypes 3^ 7 were described by Large and Sachs
in In d ia and hence used to be known as the l.a rg e -
Sachs group, T h re e further serotypes h iv e been
described making a meal o f ten.
T h is g ro u p is
named after Flexner., who de^cribtLl tht: first of the
m annito l ferm enting shigcllae from Ph ilip p in es
(19 0 0 ). T h is group is h in-h’hem ic illy li ctcrogcncnu?
and a n r ig c n k a lly the m o st c o m p le x am o n g
shigcllie. Based o r type specific and group specific
antigens, they have been cla rifie d into six serotypes
( 1 -h ) and several subtypes (la } lb ; 2 a h 2b ; 3a, .lb,
3 c n 4a, 4b, Saf 5b ). In ad d itio n , two an tig en ic
Variants' called X and Y are recognised, w hich lack
the type specific antigens. Serotype 6 is always indole
negative and occurs in three biotypes, some of w hich
form gas from sugars.
T h is group consists
o f dysentery b a c illi that resem ble S h . i J c x n t r i
biochem ically bur not aiitigenically. T h e group is
named alter B ard , w ho first described these strains
from In d ia ( 1 9 3 1 ) , Fifteen serotypes have been
id en n f ed. Sh. boydii are isolated least frequently
from cases of bacillary dysentery
T h is bacillu s, first
described by Sonne ( 1 9 1 5 ) in D enm ark, ferments
lactose and sucrose late. It is indole negative. It is
?ntigem vally homogeneous but may occur in two
forms - phase 1 and phase I I - the latter form ing
colonies that arc Larger, flatter and more irregular.
O n subculture, phase I produces both types o f
colonics but phase II is considered to be a loss
variation. O rganism s in phase II may be isolated
from patients but are mote comm on in convalescents
and carriers.
Sh. utinnti causes the mildest form o f bacillary
dysentery. In many cases the disease may o n ly be a
mild diarrhea. ] lowever, Sh. sowicd" in Lection persists
as the most com m on shigellosis in the advanced
C o p y rig h te d m aterial
countries*. For epidem iological purposes, Sh. jsamtei
has been classified into many colicifi types.
Sbijocllac cause bacillary dysentery. Infection occurs
by ingystion, The m inim um infective dose is low,
a& lew as 1 0 -1 0 0 bacilli Luring Capable o f initiating
cbe disease, preb.iblv b c c U IK they survive gastric
a cid ity better than other enterobacteria. 1 heir
p a th o g e n ic m e c h a n is m s resem b le those o f
e n te m in v a s iv e E. coli. T h e b a c illi in fe ct the
epithelial cells o f the villi in rhe large intestine and
m ultiply inside them, spreading1 laterally to involve
adjacent cells and penetrating into the lam in a
p ro p ria . I n fla m n u ru fy reactio n develo ps w ith
capillary thrombosis, leading to necrosis o f patches
u f e p ith e liu m ,‘w hich slough off, leaving behind
transverse superficial ulcers. Bacteremia may occur
in severe infections, particularly in m alnourished
children and in A I D S .
T h o u g h S h , d y s e n te riae type 1 fo rm s an
exotmbn, it appears to be m uch less important in
palhugeneHiH than the ability' of the bacillus to
p e n e tra te an d m u lt ip ly in c o lo n ic m u c o s a .
Nontoxigcnic mutants can sa il cause dysentery but
ro t nunlnvasive ones. 1 he invasive property o f the
b acillu s can he dem onstrated by its a b ility to
penetrate cultured J le L a or H e p -2 cells or by the
Congo red binding rest. Invasive property is related
to the presence in the bacillus o f large plasmids
(M .W . 14(1 x 10") coding for [he outer membrane
protein responsible for cell penetration. These
proteins are called 'virulence marker antigens'
{ V M A), Defection of V M A by I! L ib A serves as a
virulence tesr for Shigcllae, as for eaueroinvasive
Ei coH
Bacillary d y K n to y h an a sh ort i ncuhat u>T1 period
{ 1 - 7 days, usually 48 hours). 'Fhe onset and clin ical
course Hie variable and arc largely determined by
the virulence o f the infecting strain. H i e m ain
clinical features are frequent passage ot loose, scanty
fcccs co n ta in in g blood and m ucus, along w ith
abdom inal c ra m p ? and ten esm u s. Fever an d
vom iting m ay he present. Com plications are most
often seen in infection w ith Sh- dmciilcrixt: type L
and include arthritis, toxic neuritis, conjunctivitis,
p a r o tit is a n d , in c h ild r e n , in tu s s u s c e p tio n
H e m o ly t ic u re m ic s y n d r o m l- m ay o c c u r as a
com plication in severe cases. T h e severity o f the
disease may v j i t from acute fulm inating dysentery
to m ild diarrhea. A s the term bacillary dysentery
refers o n ly to the more severe cases, the term
'sh ig e llo sis’ has been em ployed to in clu d e the
whole spectrum o f disease caused by sbigeLlae.
Eluman beings arc the only natural hosts for
ehigellae. C ap tive monkeys have been found

Species Sh. dysenteric Sh. Hexneri Sh. Sordid Sh. .wnnej"

\ I j 11 II Lhl] - A A A
Lactose - - - A Late
Sucrose - - - A Late
Dulcihol - - d -

Indole d d d -
Ornithine decarboxylase - - - *
Serotypes 1(1 ft + variants li5 Only one

A - Add d - Variable

C o p y rig h te d m aterial

B io type Fermentation o f
CliiCQ ft M in n ito i
iimJ RK A A
Manchester AG AC
Newcastle- A or AG - special situations like m ental hospitals (asylum
A-Acid AG-Arid m d Gie
inferred hut such infectio ns ma y have heen o f
hum an origin. Exp erim entally dysentery c m he
produced only in monkeys, H u m an volunteer studies
.have clarified the spectrum nt shigellosis. O f a group
o f volunteers in gesting 10 ,0 0 0 Sh. flcxneri 2a
W eill], a charter remained asymptomatic, another
quarter had transient fever a day or two ]aterf a
quarter developed fever with watery djarrhea, while
onJv one quarter developed topical d i”senterv.
E p id e m ic s o f b a c illa r y
dysentery have always accompanied wars and often
influenced then outcom e. In several cam paigns,
more men have died o f dysentery than were killed
in harde, A recent instance was the major epidem ic
affecting m any thousands, with high case fatalitv,
w hich occurred during the Rwandan civil war in
1 9 9 4 , E p id e m ic s in c iv ilia n c o m Itm nities are
associated with, poverty and Sack o f sanitation.
T h e only source o f infection are hum an beings
_ cases, nr less often carriers. C h ro n ic carriage is
rare, the bacilli disappearing tram teecs w ithin <i
few weeks, except in some malnourished chidretl
or A I D S patients. bhigcllae exhibit a high rate o f
Secondary household transmission. T h e modes of
transmission may he an follows: ( 1 ) direct, through
contam inated fingers: Lh m d - to-m o u th ’ infection;
(2) through fomites such as door handles, water
taps, lavatory Stats; (3} through water; [4) through
contaminated fi>od or think. Shigeflosis, especially
Sh, snnjw i infection, may occur as food poisoning;
and {5 ) throug h flies w hich m ay transm it the
infection as mechanical vectors; (6) Shigellosis may
occur in young mate hnmwexualfi as part of the
gay bowel syndrome,
S h ig ello sis is w orldw ide in d istrib u tio n hut
epideminLogically, there are a number o f differences
lit tween the nature and extent of the infection In
the affluent and in the poor countries. W h e re
environm ental sanitation is good, as in Britain.,
shigellosis is m ainly seen in young children and in
dysentery}. Sh. inJilrtcti i£ the predominant infecting
agent. In the U S A , Sh. socmei is- the main type in
the north, while Sh. fJcxncri is more com m on in
the so u th . I n co u n trie s w here e n v iro n m e n ta l
sanitation is poor, endem ic shigellosis is found in
1 age groups and is caused by all species, Jn In dia,
Sh- tlexnert has been the predom inant species,
having formed percent o f isolates in different
series. Sh. dysenteriae ( S - 2 5 per cent) and 5fe
.mJHJICj (2™24 pet cent) are the next comm on species-
Sh. bo veil {<■>—S per cent) has been isolated least
frequently.
I he picture has changed in tecenr vears. After
a long period o f quiescence, Sh, dyreJlCH W type 1
suddenly' appeared in an extensive and virulent
epidem ic form in Central A mcrica in 19 6 8 , In 19 7 3 ,
a similar outbreak started in Bangladesh and later
in tiri Lan ka, Several localised outbreaks were
observed in In d ia from 19 7 4 , followed by extensive
epidem ics in various States from the early 1930s-
I he e p id e m ic St r a i ns -showed p la sm id borne
multiple drug resistance.
D iag n o sis is made by
isolating rhe bacillus from feces. Fresh feces should
be inoculated without delav n r transported in a
suitable medium such as Sachs' buffered glycerol
saline, p H 7 .0 -7 A H ig h lv alkaline transport media
used for vibrios are inhibitory for shigpllac. Rectal
swabs are not satisfactory.
For inoculation ir is beat to use m ucus flakes if
they are present in the sample. M a cC o n k t^ r and
D C A plates, are in o c u la te d . A fte r ovc m ig h t
incubation at 3 7 "C , the phi res arc inspected for
nonlactose fermenting colonies, w hich arc rested
for m o t ilit y and b io c h e m ic a l re a ctio n s. A n y
noTLinocile bacillus that is urease, citrate, H ,S and
C o p y rig h te d m aterial
 * tnterobacionacaae II: Shig&lla h 2B9

K C N negative should lm further investigated by w id e ly p revale nt in sh ig e lla e . In d is c r im in a t e

b io che m ical tests (Table 3 1 . 1 ) . Identificatio n is
confirm ed by slide agglutination w ith polyvalent
and monovalent sera- Dem onstration o f antibodies
in sera, is not useful.
T r e a t m e n t ; Uncomplicated shigelloNls is a self
Limited co n d itio n in w h ich the patient usually
recovers spontaneously in a few days. However, in
acute eases, p a rticu la rly in infants and yo u n g
children, dehydration has to be corrected promptly.
O ra l rehydration is adequate in most cases.
R outine antibacterial treatment is nor indicated
in dysentery- M ultiple drug resistance plasmids are
antibiotic treatment w ill only worsen the problem
o f drug resistance in intestinal bacteria. A ntibiotics
should therefore be lim ited tn severe o r tn*ie cases,
and in the very young, debilitated and the agrd.
T b e choice o f antibiotic should be b w td on the
sensii tivity o f the prevailing strai n. M a n y stra i ns are
still susceptible to nalidixic acid or norfloxacin and
other fluoroquinolones-
U o n t r o l; C o ntrol consists essentially in im proving
persona] and environmental sanitation. A ntibiotics
have no place in prophylaxis. N o effective vaccine
i-. available.

I’" Ur Lh e r H cu d in g
HaieTH. 1999. Bacillary dysentery. In T cp ley tn d W ilso n s M ic r o b io lo g y w d M ic r a b ii} I n fe c r ia n s .ty ' a in . vail. .V London:
Arnold.
Keusdi GT. 199#. Shigellosis. In Harrison's PriftiXptes ofln n rn ti Medicine. Vbl.l. 14* cdn., New York; M dlnw Hill.
LewiF MJ. 1997- Shigella- In Granwwd D {ed}. JWed.'i'fl/jVfinrh.'iiJq^y, IS^edn. Edinburgh: Church ill-L'-viqgstHK-

C o p y righted m aterial
 Enterobacteriaceae III: Salmonella
T h e p e rils SaSmonellz co n sists nt b a c illi that
p a r a s it ic the in te stin es n f a large n u m h rr of
vertebrate species and infect hum an beings, leading
to enteric fever, gastroenteritis, septicemia with or
without focal sj|)p ur«tio n, and the carrier state.
T h e most im portant member ot the genus is
Salmonella t\phi, the: causative agent ot typhoid
Fever. T h e typhoid bacillus was First observed by
Ebertb (18 8 0 ) in the mesenteric nodrs and spleen
oF Fatal cases o f typhoid fever and was isolated by
GafEky (1884). It came t^b c known as the E b e rth -
G a ffk y bacillus nr EherthcHa typhi. Salm on and
S m ith ( 1 8 3 5 ) described a b a cillu s w h ich was
believed In Cause hug cholera (m istaken!y, as !l is a
v im s d b t t t c ) .T h is hacillushlater called S. cholerac-
suis, wa^ the First of a series o f sim ilar organ isms
to be isolated from an i ria ls a ltd hum an beings -
the genus SainionvlLi. It was subsequently realised
that the typho id b acillu s also belonged to this
group, in spile of m inor biochem ical differences,
and it wm; re d e sig n a te d 5 , typhi, the genus
Ebctthclla having been abolished.
Saitnonellae currently com prise above 20l)Q
serotypes or s p e cie s. a ll o f thorn p o te n tia lly
pathogenic, Fo r practical purposes, rhoy m ay be
divided into two groups: (1) T h e enteric fever
group, consisting o f the typhoid and paratyphoid
b a c illi that are exclusively o r prim arily hum an
parasites: and (2} the food poisoning group, which
are essentially animal parasites but w hich can also
infect h um an beings, pro ducing p e tro e n te ritis,
septicemia ot localised infections.
Salmoncllac ait C r a m negative rods,
about 1-3 pm * (Li pm in sfe*. They arc motile
With peritrichatr flagella, except (or one type,
N. g-.iH inarum -puiinruni, w h ic h is alw ays
non motile. Non motile mutants o f other types may
sometimes be Found. 'ITiey do not Form capsules or
spores but m ay possess fimbriae,
S a lm o n c lla c arc
aerobic and facultalively anaerobic, grow ing readily
on sim ple m edia over a range o f p H fc-E and
temperature 1 5 - 4 1 bC {optim um 3 7 "C ).
C ol on ics arc large,. 2 - 3 m m in d iametvr, ci rctilar,
low convex and smooth. T h e y are more translucent
th an co lifo rm co lo n ie s. O n M ic C o n k e v an d
deoxycholait citrate media, colonies are colourless
due to the absence o f lactose ferm entation. O n
W ilsem and H lair bism uth sulphite m edium , jet
h i:■ l k colonies w ith a m etallic sheen arc form ed
due t< nproduction o f l 1^5. S. paratyphi A and other
species that do not form H .S p ro d uce green
colonies.
Selenile F and tetrathinnate hrcrtli are com m only
employed as enrichment media.
S alm o n cllac fo m e n t
glucu-c, [Harm - 111 ] and mall use, form ing acid and
gas. A n important exception is S. typhi w hich is
annerogciuc. Lactose, sucrose and salicin are not
fermented. Indole is not produced T h e y are M R
positive, V P negative and citrate positive. $. typhi
and a few o th e r salm o neltae d o not g ro w in
S im m o n?’ citrate m edium as they need tryptophan
as the growth factor. Urea is not hydrolysed.
is produced, except by S. paratyphi A , S. cholerae-
stds and some other jecies.
T h e enteric fever group may he separated
biochemically (Table 32,1),
C o p y rig h te d m aterial
 The: b acilli a it lulled at j 5 "C
one hour or at 60 ° C in 1 5 minute*, B o ilin g or
chlorinahon o f water and pasteurisation o f m ilk
deatniy tin- bacilli.In polluted water and soil, Utcy
survive.1 for weeks and in ire tor months. Cultures
m ay he viable for years if prevented from drying.
T h e y are killed w ithin five m inutes by m ercuric
chloride (1:5 0 0 ) or 5 % phenol,
balm oncllae possess the
following antigens based on Inch they arc classified
and identified: (1) flagellar antigen 1 1 , (2) somatic
antigen O , and (3) a surface antigen V i+ found in
so m e sp ecie s Severed strain s carry fim b ria e .
Fim brial antigens are ode important in identification
but may cause confusion due to their m ^specific
n a tu re a n d w id e sp re a d s h a rin g am o n g
ciiternbaeteriz.
T h is antigen present on the flagella
is a heat labile protein. Ir is destroyed by boiling or
by treatment w ith alcohol but not by form aldehyde.
W h e n m ix e d w ith a n tis e ra , H s u s p e n s io n s
agglutinate rapidly, prod u q rig large, Incise, Huffy
dum ps. T h e H iinfigen is strongly im m unogenic
and induces antibody formation rapidly and in high
titres follow ing infection or im m unisatio n. T h e
ilsLgdlui- antigen is o f a dnal nature, occurring in
one o f two phases.
The s o m a tic O a n tig e n is a
p h o s p h o lip id - protei n polvs acchat ide con Lplcu
w hich forms an integral part o f the cell wall. It is
identical with endotoxin. It can be extracted from
the bacterial cell by treatment w ith trichloracetic
acid, as first shown by lio iv in (atid therefore called
the Doivin antigen). Treatm ent with phenol splits
o ff ihe protein m oiety removing the antigenicity
hut retaining the toxicity o f the complex.
S. typhi A d
5, paratyphi A AG - -
S. paratyphi R AC AG
$. parafvphf C AG AG
lii T h e O antigen is unaffected by boiling* alcohol
or w eak acids, W h e n m ixed w ith antise ra, O
antigen suspensions form com pact, chalky,granular
clum ps. O agglutination takes place more slowly
and at a higher temperature optim um ( 5 0 -5 5 ^ C )
than H agglutination (3 7 ?C ) - T h e O antigen is
less im m unogenic than the 1 T antigen and the litre
o f the O a n tib o d y in d u c e d after in fe c tio n or
im m unisation is generally lower than that o f the
i l antibody.
T h c O antigen is not a single factor but a m osaic
ol two nr more antigenic factors. Salm onellac are
classified into a num ber o f groups based on the
presence ofchanacteiwtic O aitdgens on the bacterial
surface.
M a n y s tra in h o f 5. typhi fa il ro
agglutinate with the O antiserum yvhen freshly
isolated. T h is is due to the presence o f a surface
polysaccharide antigen enveloping the O antigen.
Fe lix and P itt, who first described this antigen,
believed that itw a ? related to virulence and gave it
the nam e 'V i antigen'. It is analogous to the K
antigens o f m liio r m s . It is heat labile. B a c illi
inagg hi fin ab le w irh the O antiserum h ceo m c
agglutioabk’ after boiling or heating i t 60 C for
one hour. Tt is also destroyed I tv N ! I C l and 0 5 IS"
N a O H . It in unaffected by alcohol of 0 .2 % formol.
O r ig in a lly observed in S. typhi, the Vi
antigen wirh sim ilar anrigcnic specificity is presenr
in S. paratyphi C and S. duWin, as Well as in certain
strain s o f Cdtrobdcrer (tbe B a lle r u p -E e t h e s d i
group). T h e Vri antigen tends to be lost on serial
su b cu ltu re . 1 he V i p o ly sa cch a rid e acts :lk a
virulence factor by inhibiting phagocytosis,, resisting
com plem ent activation and bacterial ly'sis by the
alternative pathway and peroxidase mediated killing.
A d
-
- ■ AG
AG -
C o p y rig h te d m aterial
In hum an volunteer exp e rim e nt, strains possessing
the V i antigen were found to cause clinical disease
more consistently [him those lacking it.
T h e Vi antigen is poorly im m unogenic and only
low rirres o f antib o dy are pro duced fo llo w ing
in fe ctio n . N o Vi a n tib o d y is in d u c e d by [he
phenolised vaccine though low titles arc produced
by' the alcoholiscci vaccine. Jl “he protective efficacy
ot [lie V i antigen is demonstrated by the success of
the purified V i vaccine tor typhoid now in routine
use. Detection o f the V i antibody is nor helpful for
the diagnosis o f cases and hence [he V i antigen is
not employed in the W id a l test. It has been stated
that the total absence o f the V I antibody in a proven
case o f typhoid fever indicates poor prognosis. T h e
antib o dy disappears early in convalescence. Its
persistence indicates the development o f the carrier
state. T h e V i a n tig e n affo rd s a m e th o d o f
epidem iological typing o f tile S. typhi Strains based
on Specific V i bacteriophages. 'M l Vrifti 'I.-!:; m Iiv.’ ■Jjtj- ; m lr*S W W (HE©.
T h e antigens o f salm o - After incubation, subcultures withdrawn from the
uellae undergo phenotypic and genotypic variations. top o f the agar outside tile central tube w ill yield a
T h is v a ria tio n is associated population o f m otile cells, {F ig , 3 2 .1} . Instead o f
w ith the loss o f flagella. W h e n saJmonellae are C r a ig ic 's tube, a U -t u b e o f soft agar m ay be
grown on agar containing phenol (liMOOJ, flagella employed, inoculation being made into one lim b
are in h ib ite d . T h is change is p h e n o typ ic and and subculture taken from the other.
temporary. Fla g e lla reappear when the strain is T h e flagellar antigens of most
su h cu ltu rcd cm m edia w itho ut phenol, Rarely, salm onelke occur in one o f two pfaflSCS, that ls, the
salm onclbic may lose their flagella by m utation. flagella m ay e x h ib it one or the o ther o f two
A stable nonmotile m utant o f i", typhi is the 9 0 1-0 alternative sets of antigens, defined hy two separate
Strain which is w idely employed for rhe preparation sets o f genes in the bacterial! pctiom e. Phase 1
o f O a g g W n a b E e bacterial suspensions. Generally, antigens arc cither specific for a sin.-cic'- or shared
the loss o f flagella is noc total and there occurs byr a few species only, f lence phase I is called the
only a dim inution in the num ber o f flagella and 'specific1 phase. Phase 1 antigens arc widely s ha red
the quantirv o f the H an tiger. Flagellated cells arc and lienee phase 2 is called the 'nonspecific’ or
found in small numbers in such cultures. T o obtain 'group' phase. Phase 1 antigens are designated a, h,
ft population o f m o n k cells, rich ill. H antigen, from e, d , etc., and after as z l , z2 , <■ tv ■ I Nba*c 2 antigens
such c u lt u r e Selection may he carried nut bv using are far fewer and arc termed 1 , 2 , etc. I n some
C r a ig ic 's tube. T h is co n s is ts of a w id e tube species, antigens belonging to phase 1 mny occur
containing soft agar (0 .2 % ) at the centre o f w hich as the phase 2 antigens (for example, I", 0, \ , z l5 ) .
is embedded a short, narrow tube open at both ends, Strains that possess both phases are called dtph.isii-.
iii such a way that it projects above the agar. Th e Som e, like 5 . typhi, occur in phase 1 only ami arc
strain is inoculated carefully into the inner tube. called m onophasic.

C o p y rig h te d m aterial
 A culture w ill contain lc ]1s with tile flagellar
antigen i; o f both phases; but generally One Or the
other phase will predominate so that the culture is
agglutinated only by one o f the phase antisera. For
s e ro ty p in g o f S a lm o n e lla iso la te s , it m ay be
nCCCt$*uy to identify the flagellar antigens; o f both
phases. A culture in phase 1 can be convened to
phase 2 by passing it through n C r a ig it's tube
co n ta in in g sp ecific phase 1 antiserum* and the
r e v e t s co n versio n achieved by u sin g phase 2
antiserum,
F re s h iso la te s o f S, typhi
generally carry a surface layer o f V i antigen that
completely masks the O antigen. Such bacilli arc
^gghitiinble w ith the V i antiserum but not with
the O antiserum. T h is is called the V form. After a
num ber o f subcultures, the V] antigen is completely
Lost. Such cultures arc inagglutinablc w ith the V i
antiserum but readily agg lutlnable w ith rhe O
antiserum. T h is Is called the W form. Intermediate
stages during the loss o f the V i Ultigen, when the
bacillus is agglutinable with both V i and O antisera,
arc called 1V W forms’.
O th e r V i-eo n rain ifig bacilli such as 5. paratyphi
C and S. dtfWiJl seld o m have the O antigen
completely masked by the V i antigen.
T h e sm o o th -to -m ug h variation
is a s so cia te d w ith the ch an g e in the co lo n y
S. analuin semtype
l.ysogenisutiort
N, ncwingtrm serotype
Lysogcnisalion
S. ininiwapolis serntype
m o rpho lo gy and loss o f the O antigen and o f
virulence. T h e colony becomes large, rough and
irre g u la r- S u s p e n s io n s in s a lin e are
autoagglutiliable. Conversion into K forms occurs
by mutation. R forms may be comm on in iaborarory
strains m aintained by serial Huhcultivation. 5 -R .
variatio n m ay be prevented to som e extent by
m aintaining cultures on Dorset's egg media In the
cold, or ideally by LyophiKsitioo.
M u c o id co lo n ie s* a s so cia te d w ith the
development o f a new m ucoid or LM ' antigen, have
been described w ith S. paratyphi B and some oilier
species.
C h a n g e s in the
structural formulae of the O antigen m ay be induced
by lym genisation w ith som e converting phages,
tcsulting in the alteration o f serotypes. T h u s , S.
anatum is converted into S. new ington by one
phage and the latter into 5 , tmnneapoUs by another
phage (Fig. 32.2).
The
classification and nomenclature o f sairnondhe have
undergone several m odificatio ns over the years.
In c lu s io n in rhe genus is based on co m m o n
biochem ical properties. C lassificatio n w ithin the
genus is on antigenic characterisation based on the
K au fS m an n -W h itc scheme. T h is scheme depends
on the id e n tifica tio n , by ag g lu tin atio n , o f the
3. 10: c, h: 1.6.
■*il.h phage 15
3. 13: e. h: 1.6.
with pilule 34
3. 15.34: c.'K; i,6.
C o p y rig h te d m aterial
 2 A S. paratyphi A 1,7,12 a -

j -B S. pariti-phi H ]. 4. 5 . 12 b 1.2
S. typhimuiitim 1.4,0,12 i 1.2
Srfhetat 4, 5 , 12 Clh tlfX
7 -C l S. pantyphi C 6, 7, (Vi) C 1.5
S. chotene-wis 6,7 c 1.5
S - C2 S. rrruerkihen 6,8 d 1.2
9- D S. typhi % 12, (Vi) d -
S. trtKfitidii 1,9,12 B-crt -

Sr gtllinanim 1, 9P 17 - -
10 - £1 Sr anstiun 3. 10 ehh 1.6

structural formulae o f the O and H antigens of the
strains {Table 32,2).
S a lm o n e lla * are in it ia lly c i t i f i e d
s e ro lo g ic a l g ro u p s, based o n the presence o f
distinctive O antigen factort, which are designated
1 , 2 t ."i, etc. Strains possessing factor 2 belong to
group A , factor 4 to group B s Filecor 9 to group D
and so on. Scrogroups were originally designated
fc» capital letters, A M»7, and a+terwards bv numhers
- currently upto 6 7. It would he more logical to
n am e the se ro g ro u p s a c c o rd in g to th e ir
chajacteristic O antigen factor numbers, rather than
by lectors. It In s therefore been pro posed to
designate group A as 2 , B as 4, C 1 ih 7, C 2 as S, D
as 9 and so on.
W ith in each group, differentiation ot serotypes
is by id e n tifica tio n o f phase 1 an d 2 flagellar
antigens. T h e K a il tt mu u n -W h it e schem e gave
species status to each serotype. T h e species were
nam ed according to the disease caused (5.ryp h i)P
the am m al source {5. gilhn^iium ), the disctuvcrer
(S. ^ o rtm u V fc ri), the name o f the. patient from
whom the first strain was. isolated {S. fhompson),,
or the place o f iso la tio n (S. poQiia). T h is was.
into four subgcnera (Table 32.3):
I, the largest and m edically t
in to im portant group contains llII the species w hich
com m only cause human and animal infections.
Submenus tl contains m ostly species isolated
from reptiles.
Subm enu* 7/jf c o n ta in s b a c illi, fo rm e rly
designated A rizona. origin ally isolated from lizards
hut subsequently found in nepedes, birds, domestic
anim als and hu m an beings. M a n y o f them arc-
prompt lactose fermenters.
S u ig rn u s I V strains arc rarely encountered and
may be considered atypical member* of subgenus 1 1.
E w in g proposed that only three species should
be recognised in the genus Salmvnella-S. L-hoierae-
StUfp £. typhi and S. cutcntidix - all other species
being considered serotypes ot S. enterititiis- T h is
proposal is now not followed.
Lactose - - + -

sarislactoiy so long as rhe teletypes W i t not t)O0 [>ulcitul 4 4 - -

d-Tartrate 4 — - -
m a n y but now w ith som e 2 4 0 0 serotypes o f
M ilonare - * 4 —

salmoncllae, giving individual names is not realistic. Sal Lein - — - +■

O n the b a s is o f b io c h e m ic a l re a ctio n s, KCN - - - +
KaufFmami proposed that salmoncllac be classified

C o p y rig h te d m aterial
 « Enterobacier ,iC'\-r ill: Salmoni.a ►
M o d e rn taxonom ical tech n iq u es, espec Lilly
D N A studies, have shown that all the members o f
the genus S.ijr.'Jo.'jf.'JjL' and o f the former genus
Arisons, me so closely related, that they should aJl
he considered as belonging to a single species, in a
g e n e tic , p h y lo g e n e tic and e v o lu tio n a ry sense.
Variations in properties such ax antigenic structure,
b io c h e m ic a l rea ct in n s an d ho st p refe re n ce s
exhibited by dliferent strains can he considered
intraspecies divergences. A new species name
i ‘. e H terii’ii has been c o in e d to in c lu d e all
salm o n ella?, S. enrertca Is classified into seven
subspecies based on D N A reussrjciatiun tests.
T h e se subspecies are nam ed enterics, salamar,
ar/zonae. didrizon&e* houtom c, bongori and in d ict
Subspecies ertrerica corresponds to the former
submenus T.
Such classificatio n and nom enclature, w hile
b e in g t u o n o m i cu lly c o rre ct, w o u ld be too
com plicated for us? in clin ical bacteriology. Fo r
example, the taaonom wally correct name for the
typ h o id bavillus w ould he *Sairrumclia. entenca,
subspecies enterics, serotype typin'. Therefore, the
old pracucc o f referring to cli-ii-- aJly im portant
sulmoncllac serotypes by the species name continues
in clin ical bacteriology.
Someli.rn.es serotype;- may have to be further
differentiated. T h u s , S. gallinaruin and S. pul/orum
cannot be distinguished semlogwaJly hut they can
be id e n t ifie d by b io c h e m ic a l re a c tio n s . (6',
U.ij'i'.'n.djirm is anaerogemc and ferments dulcitol
unlike >. pul/firum ). Im portant pathogens such is
S, iyphi, S. p-i/ityphi A and B, and S. typhimurium
can be further typed for cpidem ioiogical purposes
by phage susceptibility, biochem ical properties,
bacterociti production and ajitibiogram,
■ ‘ a ib o ^ t n ic it y t S alm o nella? are strict parasites
o f anim als or hum ans. S. typlw, S, paratyphi A and
usually, hut not in v a ria b ly S. p a ra ty p h i H are
confined to human beings. O ther salmonellae are
parasitic in various anim als - dom estic anim als,
rodents, reptiles - and birds. Som e species are host
adapted — S. .is">->ri<f’- conr found only in horses> 5,
295
ahtutus-nvis ifl sheep and lS. gul^narurr/ : il poult: v.
Others sueh as S. typhimurium, have a wide hm t
range affecting animals, birds and humans, lnfei Mon
in a n im a ls m a y vary frn m an a s y m p ta m it ic
condition to fetal, and sometimes cpi w o tic diseasc-
S. typhim unum and S. tnK ricidis cause a lata!
septicem ia in rats and m ice. S- put}arum causes
'white diarrhea' in chicks and 5 - guilinsm m fowl
typhoid.
S a lm o n e lla e cause th e fo llo w in g c lin ic a l
syndromes in human beLtigsi ( I) enteric fever
(2) septicemia, w ith or w ithout local suppurative
lesions^ and (3) gastroenteritr- or food poisoning.
ENTERIC FEVER
The term enteric fever includes typ ho id lever
caused by ft. typh; and paratyphoid fever caused by
V pamtyphi A , B and C .
Typ h o id fever was once prevalent aii h cr th ?
w orld and wax not w ell dem arcated fro:-' other
prolonged fevers. A detailed study o f the disease
wa^ p rrsc u( ed by Bretunneau (1 826) who drn 1 1 tied
the intestinal lesions. T h e name typhoid w a>given
by Lo u is (18 2 9 ) to distinguish it from typhus fever,
B u d d ( 1 8 5 6 ) pointed out that the dtsejee was
transmitted through the excreta o f patients, Eberch
(i8 6 0 ) described the typh oid bacillus and G a ffk y
118 8 4 ) j-olated it m p u r? culture. Its causative role
wax confirmed hy M etchii i knff and Besredka (1900)
hy infecting apes experimentally, A p v sty p h i A was
iso la te d b y G w y n ( 1 8 9 8 ) , S, p a n t y p h i B (S .
.scfiOttmuMcn) by A ch ard and Rcnsaude (1896 ) and
S. ptratyphi C (S. hinchfeldii) by U hlenhuth and
H u h e n c r (19 0 8 ) from cases Tescm bling typhoid
fever.
fh c infection is acquired by ingestion. In
hum an volunteer experiments, the I D 5 0 was found
to be about I f f to 10" bacilli. O n reaching th ? gut,
the bacilli attach themselves to m icrovilli o f th ?
ileal mucosa, and penetrate to the lam ina propria
and submucosa- Pher are phagocytnsed there hy
polymorph* and macrophages. T h e alulitv to resist
intracellular killin g and to m ultiply w ithin these
C o p y rig h te d m aterial
296 i Twctboofc cl Microbiology ►
cells is a measure o f their virulence. 'Ilie y enter the
mesenteric lym ph nudes, where chey m ultiply and,
via the thoracic duct, enter the bloodstream . A
transient bacterem ia fo llo w st during w h ich the
h.K'ilL are seeded. Lit the Liver, gall bladder, spleen,
bone marrow, Lymph nodes. Lungs and kidneys,
where further multiplicac ion takes place. Towards
the end o f the incubation period, there occurs
m a ssiv e bacTrrcn n .L fro m these sites of
m u ltip lic a tio n , h e ra ld in g the o nset o f c lin ic a l
disease.
A s b iiit i-s a g n o d c u l t u r e m e d i u m fu r t h e
b a cillu s, it m u ltip lies a b u n d a n tly L:i th e g all b la d d e r
and is discharged conrinuously into the in t e r n e
where ir involves rhe Feyer’s patches and lym phoid
fo llicles u f rhe ileum . T h e se becom e m il .Lined,
undergo necrosis and slough off. Leaving behind
the character-Stic typhoid ulcers. Ulceration o f the
b o w e l leads to th e tw o m a jo r c o m p lic a tio n s o f the
disease - intestinal perforation and hemorrhage,
Lh u i i it; th e 3 —4 w eek s that n o rm a lly c o n s titu te the
course o f the disease, [he intestinal lesions undergo
healing-
T h e incubation p e r ■ id is usually 7 - 1 4 days but
may range from 3 -5 6 days and appears to be related
to the dose o f infection. T h e clin ical course may
vaty from a m ild undifferentiu ted pyrexia (am bulant
typhoid) to a rapidly fatal disease. T h e onset is
usually gradual, w l:h headache, malaise, anorexia,
a coated tongue and abdom inal discom fort with
cither constipation nr diarrhea. T h e typical features
are a step-ladder pyrexia, with relative bradycardia
and toxemia. A soft, palpable spleen is a constant
finding. 1 lepatomegaly is also comm on. 'Rose spots
rhat fade on pressure appear on the skin during the
second or third week but are seldom noticeable in
dark skinned patients.
T h e most im portant com plications are intestinal
perforation, hem orrhage and cLnculalnry collapse.
Som e degree o f bronchitis or bronchopneum onia
is always found. Som e develop psychoses, deafness
or m eningitis. C h o le cy stitis, arthritis, abscesses,
pcriuslL'itii, nephritis, h e m o lytic anem ia, venous
th ro m b o ses an d p e rip h e ra l n e u ritis are o ther
com plications found. Osteom yelitis is a rare sequel.
Convalescence is slow. In about .5-10 per ce rt
o f cases, relapse occurs during convalescence- T h e
relapse rate is higher in patients treated early with
chloram phenicol ( 1 5 - 2 0 per cent).
S. paratyphi A and B cause paratyphoid fever
w h ich resembles typhoid fever b u i is generally
milder. 5. paratyphi C may also cause paratyphoid
fever but more often it leads to a frank septicem ia
with suppuradve com plications. O th e r salmonellae
h iv e on occasion been reported to cause enteric
fever. T h e se have included S. duljhu, S. baiieliy S.
sendai'. 5- enfenfitfo, -S- typhimurium, 5- castfcnume,
S. saintpaul, 5 . oran ienbtirg and S. panam a.
In fe c tio n w ith A lk a lig en es faecalis also m ay
sometimes cause a sim ilar clinical picture.
E p id e m io lo g y : Typ ho id fever has been virtually
eliminated from the advanced countries during fhe
last several d ecades m a in ly as a re su lt o f
improvements in water supply and sanitation but it
continues ro be endem ic in the poor nations of
the world. T h e control o f paratyphoid fever has
not been so s u c c e ss fu l. T h e d is t r ib u t io n o f
paratyphoid b acilli shows m arked geographical
differences. S. paratyphi A is prevalent i 1l In d ia and
other A si .in countries, Eastern Europe and South
Am erica, 5L p a rs tjjifii D in W b tc rn Europe, B ritain
and N orth Am erica; and S . paratyphi C in Eastern
Europe and G uyana.
Enteric fever is endem ic in all parts o f In dia.
A n incidence o f 980 per 10 0 ,0 0 0 was recorded in
the late 1990s in a 5-year com m unity based study
o f children in D e lh i. W orldw ide, 1 6 m illion ca^es
are estim ated to o ccu r ann u ally w ith 6 0 0 ,0 0 0
deaths! T h e proportion u f typhoid to paratyphoid
A in about 1 0 :1 . Paratyphoid B is rare and C very
rare. T h e disease occurs at all ages but is probably
most com m on in the 5 -2 0 years age group. T h e
age incidence is related to the endem icirv o f the
disease and the Level o f sanitation.
T h e source ol infection is a patient, o r far mote
frequently, a car-ifr. Patients w ho continue to shed
C o p y rig h te d m aterial
 4 E n | « r o t a c t e r i » W B III: S a l m o n e l l a
EyphniJ hacLII' in feces for three weeks To three
m onrhs after clin ical cure ire called conrafescenf
earners, T h o se who shied the bacilli for more chan
three m o n th s but less than a year are calle d
'temporary' carriers' and those w ho shed the bacilli
for over a year are called 'chronic carriers'. A bo ut
2 - 4 per cent o f patients become chronic carriers.
T h e d e ve lo p m e n t o f the carrie r state is m ore
co m m on in wom en and in the older age groups
(over 40 years). Som e persons may become earners
fo llo w in g in apparent in fe c tio n (syiriptam lefis
e x c rcto r). T h e s h e d d in g o f b a c illi is u&ua My
interm ittent.The bacilli persist in the gall bladder
or kidney and arc elim inated in the frees (fecal
carrier) or urine (u rin a ry carrier), respectively.
U rin ary carriage is less frequent and is generally
associated with some urinary lesion such as calculi
or schistosom iasis
Food handlers ur cooks who become carriers
arc parti itularly dangerous. T h e best known o f such
ty p h o id carrie rs was M a r y M a i Ion { 'T y p h o id
M jr y 'X a New York cook w ho, over a peri;>d o f I S
years, caused at least seven outbreaks affccung over
500 persons.
C a rrie rs o ccur w lrh paratyphoid b acilli also.
W h ile S. paratyphi A occurs only in hum an beings,
S, pantrypbi Q can infect anim als such as dogs or
cows, w hich may act as sources o f hum an disease.
T y p h o id fever occurs in two epidem iological
types. T h e first is endem ic or residual typhoid that
o ccu rs th ro u g h o u t the year th o u g h se a so n a l
variations may sometimes be apparent. T h e second
is epidem ic typhoid, w hich m ay occur in endem ic
or nonendemic areas. Typ h o id epidemics are water,
m ilk or foodbome.
L a b o ra to ry d ia g n o s is : B a c te r io lo g ic a l
di.ignoris o f enteric fever consists o f the isolation
o f the bacilli from the p.u i ent and the demonstration
o f antibodies in his scrum. A positive blood culture
is diagnostic, w hile the same significance cannot
be attached to is o la tio n from feces o r u rin e .
D e m o n stra tio n o f an tibodies is not co n clusive
evidence o f current infection- A third method i>
* 297
the demonstration o f typhoid bacillus antigen in
blood o r urine.
Illm id c u lt u r e : B acterem ia occurs early in the
d ise a se an d M ooli c u ltu r e s are p o s itiv e in
approximately 90 per cent o f cases in the fust week
o f fever. T h e popular belief that blood culture for
diagnosis o f typhoid fever is useful only in the Erst
w eek is erroneous. B lo o d culture is positive in
approximately 75 per cent o f cases in the second
week, 60 per cent in the third w eek and 25 per
cent Thereafter till the subsidence o f pyrexia. Blond
cultures rapidly becom e negative on treatment with
antibiotics.
A b o u t 5 - 1 0 m ! o f b lo o d Is c o lle c te d by
venepuncture and inoculated into a culture bottle
Containing 5 0 - 1 0 0 m l o f 0.5 pet cent bile hnotb.
Blood contains substances that inhibit the growth
o f the bacilli and hence it is essential that the broth
he taken in sufficient quantity to provide at least
fourfold dilution o f blood. T h e addition o fliq u o id
(sodium polyanethul sulphunate) counteracts the
bacteritiLkl action o f blood.
A fter Incubation overnight at 3 7 BC , the bile
bro th is subculturcd on M a c C o n k e y agar. Pale
nonlactose fermenting colonics that may appear on
this medium are picked out for biochem ical tests
and motility, Salmonellae w ill be m otile, indole and
urease ruga Live and ferment glucose, m annitol and
maltose but not lactose or sucrose. T h e typhoid
b acillu s w ill be in ae ro g e n ic, w h ile paratyphoid
b a c illi w ill fo rm acid, an d gas fro m su g a rs,
IdentiiidLtLoiiofthe isolate is hv slide agglu t i.nat ion.
A loopful o f the groiwth from an agar slope is
em ulsified in two drops o f saline on a slide. O n e
em ulsion acts as a control to show that the strain is
not autoagglutinable. I f S, ryphi is suspected (char
is, when no gas is farm ed from glucose), ;l loopfid
o f typhoid O antiserum (factor 9/group D ) is added
to one drop o f bacterial em ulsion on the slide, and
agglutination Looked for after ro cking the slide
gently. P ro m p t ag g lutin atio n indicates that the
i solate belongs to Salm onella group D . Its identity
jv 5 - typi'i is established by agglui inat ion w ith the
C o p y rig h te d m aterial
fffgpBar antiserum (an ti-d scrum). Q uite often, fresh
isolates of S. typhi are hi ih t V fonti and Jo not
agglutinate w ith the O antiserum. Such strains may
he tested for agglutination against an ti-V ] serum.
Alternatively, the growth h ttl^ td off in a AHUlll
am ount o f saline* boiled tor 20 m inutes and tested
for agglutination with the O antiserum. W here tlic
isolate is a noucypluud Salm onella (producing gas
from sugars), it is rested for agglutination with O
:Lfid H a n tise ra fo r g ro u p s A , B am i C . F o r
identification o f unusual serotypes, the help o f the
N atio nal Salm onella Reference Centre should be
•ought. T h e N ational Salm onella Reference Centre
Ln In d ia is located at the Central Research Institute,
KasauEi. T h e reference centre for salmon ell ,ie o f
anim al origin is at the In J u u Veterinary Research
Institute, Ir-itnugar.
I f salm oncllac are not obtained from the first
suheulmre from bile b ro ili, subcultures should be
repeated every cither day rill growth is obtained.
C u ltu re s should Ire direlared negative only after
incubation for ten days. T o elim inate [lie risk o f
in t r o d u c in g c a n t a m i n a tio n d u r in g repeated
su b cu ltu re s, and also tor eco n o m y ;tnd safety,
Castaneda’s method o f culture may be adopted, In
[his, a double medium is used. T h e bottle o f bile
b ro th bus an ig a r sla n t o n o ne R ide. A fte r
inoculation o f blood, the bottle is incubated in the
upright position. Fo r subculture* the bottle is merely
tiEte J ho that the broth runs over the surface o f the
agar. Ir is reincubated in the upright position- I f
salmoncUae ire present, colonies w ill appear on
the Riant.
A n altem ari vz to blnod culture i* rhe d o t ci 1 1turc.
H ere, 5 m l o f blood is withdrawn from the patient
into i sterile test tube and allowed to d o t. T h e
serum is pipetted off and used for the W idal test.
T h e clot is broken up with a sterile glass rod and
added to a bottle of bile broth. T h e incorporation
o f streptokinase (10 0 units per ml) in rhe broth
facilitates lysis o f rhe clot. C lo t cultures yield a
higher rate o f isolation than blood cultures as the
b a c te ric id a l a ctio n o f [he serum is o b viate d .
A no ther advantage is that a sample o f scrum also
becomes available. Ev e n though agglutinins may
be absent in the early stages o f the disease, a W id a l
test provides a baseline titnc against w hich the results
o f tests performed later m ay he evaluated.
Sal monel Inc are shed in feces
throughout die course o f the disease and even in
convalescence, w ith varying frequency. H ence fecal
cultures am almost AS valuable as blood cultures in
diagnosis. A positive fecal culture, however, may
occur ill carriers as well as in patirntfi. 'JTkc use o f
e n ric h m e n t and selective m edia and repeated
sam pling increase the rare o f isolation Fecal culture
is particularly valuable in patients on antibiotics as
rhe drug docs nor eliminate the bacilli from the
gur as rapidly as it does from the blood.
Fecal samples are plated directly on M acC onkcv,
L> C A and W ils o n -B la ir media. T h e last is highly
se le ctiv e a n d s h o u ld be p la te d h e a v ily . O n
M a cC n n k c y and O C A media, salm oncllac appear
an pale C o lc m i l- h . O n the W ils o n -B la ir m edium ,
5. typhl forms lai^e black colonies, with a metallic
sheen. .S. paratyphi A producer green colonics due
to rhe absence o f H^S productlon.
f’or enrichment, specimens arc inoculated into
one tube each o f selenite and terratliioiute broth,
and incubated tear 1 2 - 1 H hours before subculture
onto plates,
S a lm o n c lla c are shed in the
urine reregularly am ! intrequentlv. H e n ce urine
culture is less useful than the culm re o f blood
or feces. C u ltu re s are generally positive only in
rhe second and third weeks anti then only in
about per cent o f cases. Repeated sam pling
Improves the rate o f isolation. C lea n voided urine
-samples am centrifuged and the deposit inoculated
into enrichm ent and selective media as for fecal
culture.
Iso la tio n may
he obtained from several other sources but they are
not usually employed. Bone marrow culture is
valuable as it is positive in most cases even when
blood cultures are Negative- Cu lture of bile obtained
C o p y rig h te d m aterial
by duodena] aspiration is usually positive and may
be employed lor the detection o f carriers. O th er
materials which may yield isolation at times are
nine spots, pus from suppurative lesions, C.'SF and
sputum. A t autopsy, cultures may be obtained from
the gall bladder, liver, spleen and mesenteric lymph
nodes.
T h is is ,l test for the
measurement o f I I and O agglutinins for typhoid
and paratyphoid bacilli In the patient's sera. Two
types o f tubes are generally used for the test - -a
narro w tube w ith a c o n ic a l b o ttom (U rc y c ris
agglutination tulie) for the H agglutination, and a
short round bottomed tube {Fclbi tube) for the O
agglutination. Equ&l volum es (0.4 m l) o f serial
dilutions o f the scrum (from 1 / 1 0 to 1/6 4 0 ) and
the H and O antigens art mixed in D reyers .ind
Felix agglutination tubes, respectively, and incubated
in a water bath at 37 "C overnight, Some workers
recom m end incubation at 5 0 - 5 5 *C tor two hours,,
fo llo w e d by o v e rn ig h t in c u b a t io n at room
temperature. C o n tro l tubes containing the antigen
an d n o rm a l sa lin e arc set to c h e c k for
aufoaggluti nation. T h e agglutination tines o f the
serum are read. I I a g g lu tin a tio n Leads to the
formation o f loose, cotton woolly dum ps, while O
agglutination la seen as a disclike pattern at the
button] ol the tube. In both, the supernatant fluid
is tendered clear.
T h e antigens used in the test are the H and O
untigerts ol S. typin' and the H antigens of 5L p.iranphr
A and H. T h e paratyphoid O antigens arc not
employed as they CfOSs-ieacC with rhe typhoid O
antigen due to their sharing o f factor 12 . T h e I I
agglurinable suspension is prepared by adding 0 . 1 %
fo rm alin to a 2 4 - h o u r bro th cu ltu re o r saline
suspension o f an agar culture. For preparing the O
suspension,, the bacillus is cultured on phenol agar
(l:l£O 0) and [he grow th Scraped o ff in a sm a ll
volum e o f saline. It is m ixed w ith 2 0 tim es Its
volume o f absolute alcohol, heated at 4 0 -5 0 for
3 0 m i nutes, centrifuged and the deposit resuspended
in saline to the appropriate density. C h lo ro fo rm
m ay be added as a preservative. It is im portant to
use standard smooth strains for antigen preparation.
T h e strains used usually are the 5 . typJii 9 0 1, ‘O '
and 'I T strains. E a ch hatch o f antigen should he
compared with a standard. Kcadym adc W id a l kits
u f stained antigens available vuii’irncrvLidlv arc now
w idely used.
T h e re su lts o f the W jd .il test s h o u ld he
interpreted taking into account the following:
1. T h e agglutination titre will depend on the stage
o f the disease. Agglutinins usually appear by the
end o f the first week, so thnf blood taken earlier
may give a negative result. T h e tine increases
steadily till the third or the fourth week, after
w hich it declines gradually.
2. Dem onstrate >n <it'a rise in title o f antibodies, by
testing two or more serum samples* i* m ort
meaningful than a single test. I f the first sample
is taken late in the disease, a rue may not be
demonstrable. Instead, a fall in titre m ay he seen
in some caeca.
3. T h e results o f a single test should be interpreted
w ith caution. Jt is difficult to lay down levels o f
significance though it Is generally stated chat
litres ot 1 /1 0 0 Or more for O agglutinins and 1 /
30 0 or more for H agglutinins arc significant.
I l is necessary to obtain inform ation on the
distribution o f agglutinin levels in ‘normal sera‘
in different areas,
4. A gglutinins m ay be present on account o f prior
disease, inapparent infection or im m unisation,
'1'hcrefore, the mere presence o f agglutinin in
rbe W id a l test should not be taken as proof o f
ryphoid fever.
H agglutinins persist longer fban O agglutinins.
Scrum from an individual im m unised w ith "I A H
vaccine w ill generally have antibodies to 5 ,
typfii, S, paratyphi A and B , w hile in case o f
infection antibodies w ill be seen only against
tbe infecting species.
5 . P e rso n s w ho h ave bad p rio r in fe c tio n or
im m u n is a tio n m ay develop an a n a m n e stic
response during an unrelated fever. T h is may
C o p y rig h te d m aterial
300 i T a x tb o o k ; i
bcditleneirii-ited by repetition o f the test after a
wreck. T h e anam nestic response shows only a
transient r^e, while in enter! ■ fever the rise is
sustained.
6. Bacterial suspensions used as an ri.gens should be
free from fim bria. False positive results may
otherwise occur.
7. Cases treated early w ith chloram phenicol may
show a poor .ll^ltI lltLn i :i response.
O ther serological methods at diagnosis include
indirect hcmagglutinaTUm, C I E and E L I S A ,
U iIH O I^ r R A T IO N OF < iE itC U L A T iN <i
A n t ig e n
Typ h o id bacillus antigens are consis rendy present
in the blood in the early phase o f the disease, and
also in the urine o f patients, 'flu - antigen can be
d e m o n stra te d by s e n s itis e d Staph y lo c o tic il
caaggluri.na.tLon teat. Stapk. aureus (C o w an 1 strain)
w h ic h co n ta in s p ro te in A , is s ta b ilis e d w ith
formaldehyde and coated with & iyphi antihody.
W h e n a 1 % s u s p e n s io n o f s u c h s e n s itis e d
staphylococcal cells is mixed on a slide w ith seruiti
from patients in the first week o f typhoid fever, the
typhoid antigen present in the scrum combi ncs wi rh
the a n tib o d y atta ch ed to sta p h y lo co c ca l cells
produc i ng vi siblc agglut i natron wi th i n two m i nutes.
T h e test is rapid, sensitive and specific but is not
positive after the first week o f the disease.
O t h e r la b o r a t o r y te s ts i A w hite cell c o u rt
i’i useful Lcuco pcnia w ith a relative lym phocytosis
is seen. Eo sino phils are said to he absent hut lil the
tropics., w ith a h ig h in c id e n c e o f h e lm in t h ic
infestation, eosinophils arc usually present.
D ia ij n o s is o h l i u j H i i ks
T h e d e te c tio n o f c a rrie rs is im p o r ta n t fo r
e p id e m io lo g ic a l an d p u b lic h e a lth p u rp o ses.
Laboratory tests are also useful in screening food
handlers and tDoka to detect earner state.
T h e i dent ificari ■ m o f fecal carriers is by isola Mon
o f the b a c illu s fro m feces o r fro m b ile . T h e
fretpency and intensity' o f bacillary' shedding vary
W i a r a b i D lo g y ►
w idely and it is essential, therefore, to test repeated
samples. Cholagogue purgative? increase the chance
o f i^laT io n . For the detection o f u- inary earners,
repeated urine cultures should be carried out.
T h e W id a l reactio n is o f n o value in the
detection o f carriers in endem ic countries. T h e
demonstration o f V] agglutinins has been claim ed
to indicate the carrier state. W h ile Tills is useful as
a screening test, confirm ation should be made by
culture.
T h e tra c in g o f ca rrie rs in d r ie s m a y be
accomplished by the 'sewer-swab technique. G au ze
pads left m sewers and drains are cultured, and by
tracing pos i t ivc swabs, one may be led to the bouse
harbouring a carrier. Another technique o f isolating
sa lm o n clla c from sewage is filtra tio n through
nhllipore membranes and culturing the membranes
on highly selective media such as W ilso n and B la ir
media.
B a c t b r io e h a g b T y p in g
In tra s p e c ie s c la s s if ic a tio n o f S , typh i for
ep id cm io lu g ical purposes was m ade possible by
bacteriophage typing, first developed by C n ig it ; and
Yen [19 3 7 ). T h e y found that a bacteriophage acting
on the V i antigen o f the typhoid bacillus [ V i phage
II) was highly adaptable-The parent phage is called
phage A . I t could be made specific for a particular
strain o f typhoid bacillus by serial propagation in
the stra in . S u c h a d a p ta tio n w as o b ta in e d by
phenotypic or genotypic variation. A t present, 9 7
V i I I phage types o f S, ryphi are recognised. A s
phage typing o f S. n y hi depends on the presence
o f V i an ri gens, a proportion o f strains ( V i negative)
w ill be untypable. T h e phage type is stable. A p art
from helping in tracing the source o f epidem ics,
phage typ in g also provides inform ation cm the
trends and patterns in the epidem iology o f typhoid
at the local, national and international levels. Phage
typing is carried out at the N atio n al Phage T yp in g
C e n tre s and is coordinated by the Internatio nal
Reference Centre. T h e N ational Salm onella Phage
T y p in g C e n tre for In d ia is located at the L a d y
C o p y rig h te d m aterial
H a rd in g c M e d ica l C o lle g e , N ew D e lh i, Phage various parts o f E u ro p e , A frica and A sia where
types A and E l arc the moRt com m on and are typhoid and paratyphoid fever? were endem ic. N o
present throughout India. How ever, the relative controlled field trial has been conducted w ith the
prevalence in different regions in subiect to change T A B vaccLm.'. In civ ilia n p ractice, protection is
from tim e tn lim c. m ainly required against typhoid fever. I f paratyphoid
The preponderance o f one or two phage types components arc considered necessary cither A or
in a region limits the utility o f phage typing as H m ay be added, but nor both, as only one o f them
an epidem iological tool, A d d itio n a l markers is found in any one area,^Therefore, in In d ia, instead
have, therefore, been employed for the subdivision ui [h e T A B vaccine, a divalent typhoid—paratyphoid
o f strains belonging to a phage type. These include A vaccine {elim inating paratyphoid B w hich is Very
(1) N ir d lh 'i complementary phage typing o f rare in the country) or the monovalent typhoid
type A strain s into 10 types; (2 ) K r is te n s c n s vaccine is preferred-
biotyping based on ferm entation o f xylose and T h e vaccine is given in two doses o f 0 .5 m l
arabtnose; (3) production o f tetrathionate reductase, subcutaneously at an interval o f 4 -6 weeks. Lo ca l
(4) bacteriocin production; and (5) antibiogram . and general reactions lasting for one or two days
C u rre n tly , more d is c rim in a tin g ge n o typ in g are quite frequent. Such reactions may be avoided
methods like phiRmid finger printing, multilnCMS if tbc vaccine is adm inistered in a dose o f 0 .1 ml
enzym e e lectro p h o re sis, IS -2 G Q p ro filin g and Intraderm ally In nonendcm ic areas, vaccination is
random am plified polym orphic D N A analysis, have recommended for troops, m edical and paramedical
been employed for epidem iological characterisation p e rs o n n e l. I n e n d e m ic areas v a c c in a t io n is
in advanced centres. recom m ended for all children, in whom a sin
Phage typing has been applied also tn S. paratyphi dose m ight give adequate protection, which m ay
A and B , S. typhimurium, S. cntcritidia and 5,
tJub/rih A m o n g [lie 5. pararyphr A isolated from
In d ia, phage types 1 and 2 are the most common.
T y p h o id fever can he effectively
c o n tr o lle d by g e n e ra l m e asu re s, su ch as
im provem ents in sanitation and provision o f
protected water supply. M a n y developed countries
have been able to e lim in a te the risk by these
measures, but occasional outbreaks do appear due
to unforeseen lapses.
Specific prophylaxis w ith beat killed typhoid
bacillus vaccine was developed and succu sstully held
tested by A im rath W rig h t during the Hner war in
South Africa, T h e T A B vaccine w hich came into
general use larer contained S. iyphit 10 0 0 million
and 5, paratyphi A and B , 7 5 0 m illion each per ml
killed by heating at 5 0 -6 0 "C and preserved in
0 ,5 'W phenol.
T h e use o f po lyvalent T A B vaccine w as an
accident o f history. It was introduced in that form
in W nrld W ar I , as British troops bad to serve in

C o p y rig h te d m aterial
be m aintained fttf several yean-H y rhc booster effect
o f repeated natural sub dinical infections.
'IV ip new typhoid vaccines, one oisil and the
o th e r in je c t a b le , h ave heen in tro d u c e d after
successful field trials. T h e live oral vaccine (lypJiora/)
if a stable m utant o f 5, tYplti Strain T y 2 la , lacking
the enzym e lJ D P 'g 9 lK t o s c -4 -t p im t n it fG a l F.
mutant). O n ingestion, it initiates in t e r io r but 'self
destructs' after four o r five cell d iv isio n s , and
therefore cannot induce any ill ness. T h e vaccine lh
an enteric coated capsiute co n ta in in g 1 0 ’ viable
lyophilised mutant bacilli. T h e course ccinsists o f
one capsule oraUy, taken an hour before food, with
□ glass o f w ater or m ilk, tin days 1, .1 and 5. N o
antibiotic should be taken during this period.
The injectable vaccine (typhim *Vi) contains
purified V ] polysaccharide antigen (25 pg per dose)
from S. ivphi strain T y 2 . I[ is given as a single
subcutaneous or intram uscular in jectio n , w hich
causes only m inim al local reaction.
Both these vaccines are recommended only in
chose o i l ]1 five sx-arv oJ" age, the same dose being
u sed fo r c h ild r e n and a d u lts. In b o th cases
protection is stated tci com m ence 2 - 3 weeks after
adm i lustration and lasts for a r least three ye ais, after
w hich a h<UKlcr may be given. Roth the vaccines
are effective and only their relatively high cost stands
in the way o f their w ider use.
T y p h o id b a c illi are p r im a r ily in trace llu l.tr
parasites, and cell mediated im m unity rather than
h u m o ra l a n tib o d ie s m ay he m ore relevant in
p ro te ctio n ag a in st [hr disc use. C e ll m ediated
im m un ity develops during the course of" [he disease.
C e llu la r i m m u n i tv to the typ h o id b a c illu s is
com m on in populations in endem ic areas. Absence
o f C M I hashecn claim ed to indicate susceptibility.
T h e killed vaccines currently uned do not stimulate
C M I.
S p e cific a n tib acte rial therapy for
enteric fever became available only in 19 4 S w ith
the in t r o d u c t io n of c h lo r a m p h e n ic o l, w h ic h
continued us the sheet anchor iigninsl the disease
till the 19 70 s when resistance became com m on.
Though S. ryphi is susceptible in vitro to many
antibiotics such as streptom ycin and tetracycline,
these arc ineffective in vivo. A m p icillin , amoxycillin,
furazolidone, and cotriiTiEixuzEjle were the other
drugs that had been found useful in the treatment
of typhoid fever.
W h ile antibacterial therapy has been so effective
in the treatment o f cases, it has b « n disappointing
l11 the treatm ent o f carriers. A combination o f
anlihactcrial [herapy along with flic vaccine has
been tried in the eradication o f carrier state, ^ “his
com bination has also been used to prevent relapses.
Elimination o f the carrier state may require heroic
measures such as choLcc^ tectomy, pyelolithotom y
or nephrectomy.
T h o u g h occasional resistant
stra in s had been id e n tifie d in the laboratory,
resistance to chloram phenico l d id not pose any
problem in typhoid fever till 1 9 7 2 , when resistant
Strains emerged in M exico and ill Kerala ! India).
Tn M exico, the resistant strain caused an explosive
epidem ic, w ith high mortality. Travellers w ho got
infected in M exico had, on occasion, conveyed the
resistant strain to N orth A m e rica and Eu ro p e but
Lt d id iH»[ get e s ta b lis h e d in these areas.
C h lo r a m p h e n ic o l re sistan t ty p h o id fever has
become a problem in many countries in A sia,
In India, chloramphenicol resists or typhoid fever
appeared in epidem ic form first in C a licu t (K e ra k )
m early 19 7 2 . Jt became endem ic and was confined
tu K erala till 197-H. S u b se q u e n tly such strain s
carrying drug resistance plasm ids appeared in m any
O ther p a rts of I n d ia . T h o u g h re s is ta n t to
c h lo r a m p h e n ic o l, s u c h stra in s w ere in it ia lly
sensitive to atnpicillin, am oxycillin, eotrim ojiaxok
and furazolidone, which were successfullv used for
treatment. B v late 19ftDs, Typhoid bacillus Strains
resistant to m any or all o f these drugs began to
spread lei most parts o f India. A r present, the drugs
useful in treatm ent of such multi resistant typhoid
cases arc the later flu o ro q u in o lo n e s (su ch as
ciprofloxacin, pcfloxacin, ofloxacin] and the third
generation cephalosporins fsuch as ceftazidim e.
C o p y rig h te d m aterial
eeftrioxone, cefotaxime), Furo^olidonc if still active
against most isolates but its action is too slow for it
Co be used alone in treatment. Recently many strains
have becom e resistant to fluoroquinolones, but
several isolates o f typhoid bacilli are now seoisitive
co cldoram phenicol.
S a lm o n e lla gastroenteritis (m ore appropriately
e n tero co litis) or food p o iso n in g is generally a
zo o n o tic disease, Che source o f infectio n being
anim al products. It may be caused by any salmonella
except S, typhi. T h e first instance o f salmonella food
po iso n in g to have been identified was in 18 S 8 ,
when G ie rtn e r in G erm an y isolated a bacillus (i'.
e n tcr/fj'd if) from th e mejit o f an e m e rg e n c y '
slaughtered cow and from the cadaver o f a fatal
ease o f food poisoning caused by the meat. In 18 9 8 ,
D u rh am in E n g la n d and de Nobele in B elgium
isolated S. typhimurmm from meat and from food
poisoning cases. A v e ry large number ofsalin o u d lac
h ave s in c e been id e n t ifie d fro m cases o f
gastroentc] itis and food po iso n in g but a lew
species account for the majority o f cases. In most
parrs o f the worlds S . typhim urium is the moat
com m on species. Som e other com m on species have
been S. entcritidis, S. ha/Jan .!>, Heidelberg, 5. agoni,
S. vitchuwT S, se/renAcrif, 5. indiaitaf S. ntw pori
and S- yn^tvm-
H u m a n Infection results fioiri the ingestion o f
contaminated food. T h e most frequent sources o f
salm onella food poisoning are poultry; meat, m ilk
and m ilk products. O f great concern are eggs and
egg products, Salm onella^ can enter through the
shell if eggs arc left on contaminated chicken feed
or feces, and grow inside. H u m a n carriers do occur
h ut th e ir role is m in im a l w hen co n sid ered in
relation to the magnitude o f infection from animals.
E v e n salads and other uncooked vegetables may
cause infection if contaminate J through manure Of
by handling. Pood contamination m ay also result
from the droppings o f rats, tixards ot other small
anim als. Gastroenteritis m ay occur w ithout fond
poisoning ns in cross infection in hospital*.
C lin ic a lly , the disease develops after <t short
Incubation period of 2*1 hours or less, w ith diarrhea*
vomicing, abdom inal pain and fever. It m ay vary in
severity from the passage o f one or two loose stools
to an acute cholera-like disease. It usually subsides
in 2 -4 days bur in some cases a more prolonged
enteritis develops, with passage o f m ucus und pus
in feces, resembling dysentery. In a few, typhoids!
or septicemic type o f fever may develop.
Laboratory diagnosis is made by isolating the
salm onella from the feces* In outbreaks o f food
poisoning, the causative article o f food can often
be identified by taking a proper history. Isolation
o f sal m nnellse from the article o f food confirm s
the diagnosis.
C o n tro l o f salmonella food poisoning requires
rhe prevention o f food contam ination. Food may
become contaminated ar various levels* from natural
infection in the an Lina] ot bird, to contam ination o f
the prepared food, Proper cooking o f food destroys
saJmonellae.
W h ile enteric fever is a major problem only in
the developing countries, salmonella food poisoning
is largely a problem for the developed nations. "Iliis
is due ro rhe differences in food habits and living
conditions between them and also because food
production, piickagfog, s-tu-rage and m irk e tiiig have
become industries in the rich countries w hile they
still remain agricultural in the developing world.
fre a tm e n t o f u nco m pl icate d , n-m t in v a »ive
salm onellosis is sym ptom atic. A n tib io tics should
nor be used. N o t only do they not hasten recovery
but they m ay actually increase the period o f focal
shedding o f the b acilli. But for the serious invasive
cases, antibiotic treatment is needed.
C e rtain salmonellac* >, dtoferaesuis in particular,
mav cause septicemic disease with focal suppurative
lesio n s, su ch as o ste o m yelitis, deep abscesses,
C o p y rig h te d m aterial
304 'i Tarttxnk of Vmroo dogy *

endocardltis, pneumon 1 1 and mem ngiti^, Antecedent
gastroenteritis m ay or may not he present. T h e case
fatality may be as hii.ith as 25 per cent.
Salm oncllac may be isolated from the blood or
from the pus from the suppurative lesion. Feces
culture m ay also sometimes be positive. Septicem ic
s a lm o n e llo s is sh o u ld be treated w ith
d ilo ram p h cn ito l or -other apprupn.Lte antibiotics
as determined by sensitivity tests.
MULTI RESISTAN T SALM O NELLAE
R factors conferring m ultiple drug resistance have
become w idely dissem inated am ong salmonellae.
T h e clinical significance o f t! i ls phenomenon was
first observed during the studies o f hum an and
v e te rin a ry in fe c tio n s w ith d ru g re s is ra n r
Srtyphifnurwni phage type 29 in En g lan d in the
l'VfiOs. H u m a n infections were in itia lly gastno-
erteri tis due to spread from i ufccted an imats, through
food. Subsequently some salmoneUac appear to have
changed their ecology in some ways. Fro m being
responsible for zoonotic infections only as in the past,
some multiresisiant s&lmonellae have now become
important agents o f huspit.Ll cross infections. Soicti
nosocomial salmonellosis manifests particularly in
neonates as septicemia, meningitis and suppurative
lesions. Diarrhea may not always be present.
I n lndia> several hospital outbreaks o f neonatal
septicem ia caused by m ulti resistant salm oncllae
have occurred in recent years. M ortality in neonates
is. veTy high unless early treatment is started w ith
andbiotics to w hich the infecting strain Is sensitive.

F u r t h e r R e a d in g
Christie AJ1. I9H7. Inicciious Disease*: Epidcmiahtgy ,n.;S C.'Itnii .d JV.-j. .■nl-. 411«dn. Edinburgh: ChiLruhill-Lhin^tvnc.
Forayth JHL. 199EJ. Typhoid\tndPariryphoid. In Topicy -md Wllsfuis Aftcfobiuk^y and JVJjftYfibra/ Jn&.'firirwh9* nlil- mi-3,
ljurulun: A: ik:U.
Ivrnnrt Hc( ai-! 994. Vaocination against typhoid t'ever Present jrJP-i--, ftrJJ W ki Hlrti Opp 72^957-
AEj.ikLl'1 RK. 1994. S:l1:11111.-1L.i rypltii mill citIiet -_11r.i■ri■:-!I.l- ( J:;t i'i:7]-:v
MitiaSH er iL 1996. MuJriiim^ ft.-i-.iiiu Silmerttlli typhi A pivbicift - J M knobioi. 44:317.
W HO, 1994. C’Hwitml nf !-..iI:i: -1l I' :I infection* in jnim ilL, ftu lf Wlrf Ifith flrg , 7^. 11,

C o p y rig h te d m aterial
 Vibrio

V ibrio * arc G ra m negative, rigid, curved rods that o f 16-40 °C (optimum 37 °C). Growth is better in
arc actively motile by means o f a polar flagellum. art alkaline medium the ran g eo fpH being ft.4—'9 .6
T h e name 'vibrio' in derived from the characteristic (optim um 3 .2 ) . N a C l ( 0 . 5 - 1 % ) is required for
vibratory m otility [from vibrate, meaning to vibrate). optimal growth though high concentrations (6%
T h e y are asporogenous and noncapsulated. V ibrios and above) are inhibitory.
arc present in m arine environm ents and surface It grows well on ordinary media. O n nutrient
wafers worldwide. T h e most important member o f agar, after overnight growth, colonies are m oist,
the genus is Vibrio choleras, the causative agent o f translucent, round discs, about 1 - 2 mm in diameter,
cholera. It was first isolated by K o ch (ItfB l) from with i bluish tinge in transmitted lig h t-"iT c growth
cholera patients in E g yp t, though it had been has a distinctive odour. O n M a c C u n k e p ig u , the
observed earlier by P acin i (11354) and others. colonies are colourless at first but become reddish
o n p ro lo n g e d in c u b a t io n due to the late
. ___ ________ 1 — ______L —_ » _ J l. .
fermentation o f lactose. O n blood agar, colonies
: T h e ch o le ra v ib rio is a s h o rt, ate in itia lly surrounded by a zone o f greening.
curved, cy lin d rica l rod, about 1 .5 pm * 0 .2 -Q .4
pm in S tic, w ith rounded Of slightly pointed ends.
T h e cell is typically comm a shaped (hence the old
name V. com m a) but the curvature is often on
subculture. S-shaped or spiral forms m ay be seen
d u e to two nr m o re c e lls ly in g en d to end.
Pleom oiphism is frenuent in o ld -cultures. In stained
film s o f mucous flakes from acute cholera eases,
the vibrio s arc seen arranged in p arallel row s,
described by K o ch as the 'fish m stream' appearance.
It is actively motile, with a single sheathed polar
flagellum. T h e m otility is o f the d u rin g ty p ^ and
w hen acute cholera stool nr i young culture is
exam ined under the microscope, the actively motile
vibrios suggest a 'swarm o f gnats'. T h e vibrios stain
readily with aniline dyes and are G ra m negative
and nunacid fast (Fig. 3 3 ,1).
r: T h e cholera vibrio
is strongly aerobic, growth being scanty and slow
an aerobically it glows within j temperature range

Copyrighted m atari
w hich later becomes 4 cm " due hemodigestion.
In g ck rin stab culture, Lnfim ditm lifarai tfoim ef-
shujted) «r nupitnrm (turnip shaped) liquefaction
occurs in three days at 21 *C , In peptone water,
growth occurs in about six hours as a fine surface
p e llic le , w h ic h o n s h a k in g b reaks up in to
m em branous pieces, T u r b id ity and a pow dery
deposit develop on continued incubation.
A num ber o f special media h iv e been employed
fuf the cultivation n f cholera vibrios. T h e y may he
classified .ls follows:
(1) V e n L n -
n im a n -R jm a k ris h n -a n ( V R } m edium : A sim ple
m odified form o f th is m e d iu m is prepared by
dissolving .20 g crude sea salt and S g peptone in
one litre o f distilled wafer and adjusting the p H to
K.fv-S.S. Jt La dispensed in screwcsppcd bottles in
1 0 - 1 ^ ml am ounts, A bo ut 1 - 3 ml stool is to be
added to each bottle. In this medium vibrios do
not m ultiply but rem ain viable for several weeks.
(3) ( T r y —Rluir m edium: T h is is a buffered solution
o f s o d iu m c h lo r id e , sorfium t lu o g lv c o ll ate,
d isodium phosphate and calcium chloride at p H
8,4. It is a suitable transport m edium for Salm onella
and Shigella us well as tor vibrios. (.1) Autoclaved
sea water also serves ns ;i holding m edium .
(1) Alkaline peptone water
at p H tf.6; [2 ) M o n s u r ’s taUrncholate tellu rite
peptone water at p H 9 .2 . B oth these arc good
transport is well as enrichm ent media.
(1) Alkaline bile salt agar (E S A )
p H 8,2: T h is Minple medium bus stxHid the test of
time and is still widely used-The colonies are simitar
to those on nutrient agar. (2 ) M o n s u rs gelatin
taurodiolate trypticase tellu rite i^^r ( G I T A j medium r
Cholera vihriim pmdm X small, rr.msluccnt colonics
with a greyish black centre and a turbid halo. T h e
colonies become 3 H min in siae in 48 hours. (3)
rCBS medBwnul bis; medium, contain ipg thiosulfate,
citrate, bile salts and sucrose, is available commercially
and is very w idely used at present. Cholera vibrios
produce large yellow convex colonies which may
become green on continued intubation.
V ib rio colonies m aybe identified by the 'string
test'. A Loopful o f the growth is mixed with a drnp
o f 0 . ? % sodium dcoxycholiite in saline on a slide.
I f the test is p o sitive, the suspensio n loses its
turbidity; becomes m ucoid and forms a 'string'when
tin; loop is drawn slowly away from the suspension.
C a r b o h y d r a te
m eialm lism is fermentative, producing acid, but no
gas. Cholera vibrios ferment glucose, mannitol,
maltose, m nr nose and sucrose but nor inositol,
arabinosc, or lactose, though lactose may be split
very slow ly, In d o le is form ed and nitrates arc
reduced to nitrites. Th e se two properties contribute
to the 'cholera red reaction' w hich is tested by
adding a few drops o f concentrated sulphuric acid
to a 2 4 -hour peprone water culture. W ith cholera
vibrios, a reddish pink colour is developed due to
the fo rm atio n o f n it r o s o -indole. C a talase and
oxidase tests ,ire positive. M eth yl red and urease
tests are negative. V ibrio s dccarboxyUte lysine and
ornithine but do not utilise arginine. G e la tin is
liq u e fie d . V ib r io s elab o rate several en zy m e s
in c lu d in g e a lla g t n is e , e la sta se , c h it m u e ,
nucleotidase, decarboxylase, lipase1, mueinasc and
neuraminidase (receptor destroying enzym e).
C h o le ra vibrios are 'susceptible to
beat, drying ar.d acids, but resist high alkalinity.
T h e y arc destroyed at 55 UC in 15 minutes. D ried
on linen or thread, they survivt for 1 - 3 days but
die in about three hours o r covef slips. Survival ill
water is influenced by irs p H . temperature, salinity,
presence o f organic pollution and other factors, In
general, the E l T o r vibrio survives Longer than the
classical cholera vibrio, In the laboratory, vibrios
survive tor months in sterile sea water, and this has
been suggested as a method for the survival o f vibrios
]]~l nature. Ill gro&lv CCnturuinated water, such as the
Ganges water ot India, the vihiiof do not survive for
any Ifll Igth oft ii IK , due to [he i | ^patently large amounts
o f vibrio phages p it sent. T h e y survive in dean tap
water fiw thirty days. In untreated night soil, they may
survive for several days. Vibrios are susceptible to the
common disinfectants.
C o p y rig h te d m aterial
 O n fruits, they survive for 1 -S days at room
temperature and for a week in the refrigerator. In
general, food materials left at room temperature do
ro t act as an important KHim eof irfc jtio n for longer
than a day o r two but those stored in the cold may
harbour vibrios for more than two weeks.
T h e y are killed in a few m inutes in the
gastric juice nt’ normal acidity but they may survive
for 24 hours in achlorhydric gastric juice.
Jn the p astT m a n y o x id a se
positive, m otile, curved nods were rather loosely
grouped as vibrios. Precise criteria have been laid
down for differentiating vibrios from related genera
(T ab le 3 3 .1 ) .
H eiberg (19 3 4 ) classified vibrios into six groups
based on the fermentation o f mannose, sucrose and
arubinose- Tw o m ore groups were added later,
Ch o lera vibrios belong to C r o u p I (Table 33.2).
A serologies! classification was introduced hv
G ardner and Venkxtruman (19 3 5 ). C h o lera vibrios
and b io ch e m ica lly sim ilar vibrios, possessing a
com m on flagellar ( H ) antigen were classified as
G ro u p A vibrios, and the rest as G ro u p B vibrios
com prising a heterogeneous collection. Based on
the major somatic (O ) antigen. G ro u p A vibrios
were classified into ^subgroups' (now called t>
serogroups Or seruvaisX 3 39 o f w hich are currently
know n (Tab le 3 3 ,3 ), A l l isolates from epidem ic
cholera (till 19 9 2 ) belonged to semigroup 0 - 1 .
Therefore in the diagnostic laboratory group 0 - 1
a n tis e ru m (c o m m o n ly c a lle d "cho lera
n o a d if f e r c n t ia l seru m ') cam e to be used for
identifying pathogenic cholera v ib r io (which are
referred to as ‘agglutiiiabk vibrio*'), O ther vibrio
isokces w hich were not agglutinated by the 0 - 1
.antiserum. came to be called nonaggiu tiiiiiife or
M A G vibrios, rrh cv were considered nonpathogenic
and hence also called noncholcra vibrios ( N C V ) .
B oth these terms me not Strictly appropriate.
T h o u g h N A G vibrios are not agglutinable by the
0 - 1 antiserum, they are readily agglutinated by their
own antisera. T h e term noncholera vibrio is no !
OOmqct as spm eot rhe-nn can cause a disease clinically
indistinguishable from cholera, How ever, by and
larg e, M A G v ih rio s were non p a th o g e n ic an d
com m only isolated from environmental sources and
healthy hum an intestmes-
W h il e a ll iso late s fro m e p id e n tic ch o le ra
belonged to group 0 - 1 , not all members o f the
group Were capable o f c.iusing clinical cholera. T h e
first such m embers w hich acquired prom inence
were the vibrios isolated by G o tls c h lic h |1 9 0 ? )
fro m six H a j p ilg r im s w ho d ie d at the T o r
quarantine station on the Sirtaj Peninsula.T h e y had
d ied not fro m ch o le ra but from d ysen tery or
gangrene o f the colon. T h e se came to be called the
E l lo r v ib rio s. T h e y were id e n tical to cholera
vibrios in all laboratory tests except that they were
h em o htic to sheep erythrocytes and gave a positive
^ogcs-Pnoskaner reaction, A s the E l l o r vibrios
were subsequently isolated from water sources and
normal humxm intestines, they were considered to
be nonpathogenic. In 19 3 7 , E l Tor vibrio s were
reco g n ise d as en d em ic in C e le b e s (S u law e si),
Indonesia, causing a choleraic disease (paracholera).
I iowever, outside this endem ic area, E l l o r vibrios
were considered nonpathogenic.
T h e situation changed in 1 9 6 1 when E l For
v ib rio s gave rise to the seventh p a n d e m ic n f
c h o le ra , b e s id e s in cre a se d v iru le n c e and
lnvaiiveness, the pandem ic E l T o r strains showed
altered laboratory reactions. T h e new E l T o r strains
were often nonhem olytic and V —F negative, New
differed haring properties were defined such as chick
cell agglutination, polymyxin sensitivity and phage
su^rep tihilily tests (Table 33.4 ). It was accepted
that E l T o r vibrios were indeed cholera vibrios,
w hich contained two bioiypeSf the old or 'classical'
cholera vibrios, and the E l To t vibrios.
Based on m inor surface antigen ic characteristics
both classical and E l T o r biotypes o f cholera vibrios
were classified into three serotypes, Ogaw a, Inaba,
and H ik o jim a (Table 3 3 ,5 ). T h e Oguwa and Inaba
strains are agglutinated by their nwn respective
specific seta only, w liik the I liko jim a itrains arc
agglutinated hy both Q gaw a and Inaba antisera.
C o p y rig h te d m aterial
Vibrio * 41 +■ f +■
A eium unis *■ *2 - + - V
Pseudomonas + - V V V -
P lc K litH IW H ia S 4- -4 4- + 1- | -

Note : 1 * no gas produced' 2 » gas mayor may nor be produced, V = reaction varisble.

T h e re is no difference in pathogenicity between Further classification ca r be made by phage

th e three se ro ty p e s. S c r o t y p in g is o n ly o f
ejudemiulogLCal significance.
T h e n o n -0 1 vibrios (ilie so culled N A G vibrios)
have been classified into m any serogroupi, currently
upIO lily . T h e latest serogroup O -t .1 9 , identified
in 1992 causes epidemics o f cholera, em phasising
that they can no longer he considered as noncholera
vibrios.
M o d e rn taxonom ic a l c rirc ria h p articularly
D N A studies, have led to the recognition that all
the cholera vibrio s rhat belong to G a rd n e r and
V'enkatram am 's g ro u p A an d sh are s im ila r
biocheniLLal properties and a comm on H antigen
arc so clo sd y related that they constitute a single
species Vibrio cholerae, w hich can be classified
into serognciups (urseruvars), bin types :md scrotypes.
A c c o rd in g ly the present nom enclature w ill he
indicative o f all these features, as for example,
VT cholcrat scrovar 0 1 , biotype E l Tor, serotype
Ogawa.
t y p in g . Phage ty p in g sch e m e s h ave been
standardised for classical and E l Tor Iriotypes an as
well as for 0 - 1 1 9 vibrios. New molecular methods
like ribntyping have added further refinements to
strain typing.
Cholera is an acute diarrheal disease caused by
V. tholcntc. In its most severe lbrm f cholera is a
dram atic and terrifying illness in w hich profuse
painless w atery diarrhea and copious effortless
vom iting may lead to hypovolemic shock and death
Lit less than 2 4 hours. In created canes, the disease
may last 4 -fi days, during w hich period the patient
may pass a total volum e o f liquid stool equal iu
twice bis body weight. A ll the clinical features o f
severe cholera result from this massive loss o f fluid
and eJecirdytfts- T h e cholera stool is typically a
rolouricss waterv fluid w nh flecks o f m ucus, said
to resemble water in w hich rice has been washed

T A A -

11 - A -

[11 A A A
IV -
A A
V A - -

VI - - -

V ll A - A
VHI - —
A

C o p y rig h te d m aterial

Croup A
Cholera vibrios
!li:■■ hetmcal similarities;
Common H anrigen
{ Vihfirt ^tialera?)
O SUBGROUPS
(SEROGROUPS) :
SEROVARS)
01
BlOTYl’ES 'I
[Crireria Listed K
in Table 33-4) J
‘ 1
SERO TYPES Oi\.m.i 111.i':m
TiWie 33,3 Ginfcw «rosl Vm**tnnw'* tlWWtVWfl (updatedi
{hence called 'rice water s too Is ). It has a
characteristic inoffensive sweetish odour. Tn
composition it is a bicarbonate-rich isotonic
electro vie solution, with Lirtle protein, Its
outpouring lead ■,to diminu t iim of cxtracdlular fluid
volume, hemoconcentratioriH hypokalemia, base-
deficit acidosis and shock. The common
complications are muscular cramps, renal failure,
pulmonary edema, cardiac arrhythr iias and paralyl iC
ileus. The clinical severity of cholera varies widely*
from the rapidly fatal disease to a transient
asymptomatic colonisation of the intestine by the
vibrios* The incidence of mild and asymptomatic
infections is more with El Tor vibrios than with
the classical cholera vibrios.
The incubation period varies from less than
24 hours to about five days. The clinical illness
■*. Vibrio ► 109
VIBRIO
Group B
JH
-Li .1|]y and
antiger1ically
heterogeneous
Non-Ql {cunently upto 0-13)
Hiktuima
may begin slowly With mild diarrhea and vomiting
in 1-3 days or abruptly with sudden massive
diarrhea.
Futhogcueflis: Natural infection with cholera
occurs only in humans and not in animals. A number
of animal models have been developed whida have
helped in understanding the pathogenic
mechanisms in cholera. The first of these was the
rabbit ileal loop model of De and Chattedcc { ^953).
1nicerion of cholera culture or culture filtrate into
the Ligated iIeal loop caused fluid aceumulalion and
ballooning. Intestinal loops of many other animals
and also of ehickens have been shown to behave in
a similar manure Dutta and Habbu Cl 955) showed
that a fatal diarrhea could be induced ir infant
rabbits infected with \ibi 10 a perorally or
intr|Lintestinally. Sack and Carpenter {19661
C o p y rig h te d m aterial
introduced the canine model. C ogs mfcewd with
cholera vfbrios through a stomach tube, after
administration of sodium bicarbonate developed
diarrhea and vomiting* usually ending in death.
In human infection, the vibrios enter orally Hemolysis -

Vbges-Praskauer +*
through contaminated water or Food- Vibrios are
Chi d: erythrocyte — J t

highly susceptible to acids, and gastric acidity agRjimntfinn

provides ail effective barrier again^r s-niill doses of
cholera vibrios. It has been shown that 1114
pathogenic vibrios administered to fasting
n o rm o ch lo rh vdric volunleers* ■without food or
buffer, did not produce infection, while the same
dose given along with food or sodium bicarbonate
caused clinical cholera itl 80 100 pet cent of them.
Achlorhydria predisposes to cholera in the field.
In the small intestine, vibrios arc enabled to cross
the pnoftethe layer of mucus and reach the epithelial
cells by chcmotaxis, motility, itiucinase and other
proteolytic enzymes. A hemagglutinin-protease
(formerly known as 'cholera lectin') cleaves mucus
and tihrorcclin. It also hi/lps in releasing vibrios
hound t4x bowel rtrUcosa, facilitating their spread to
other parts of the Intestine and also their fecal
shedding. Adhesion to tire epithelial surface and
colonisation may he facilitated by special fimbria
such as the 'toxin co-regulated pi Ins' ( [ C P).
Throughout the course of infection, the vibrios
remain attached to the epithelium hut do not
damage or invade the cells. The changes induced
are biochemical rather than bistological-
Vibrios multiplying on the intestinal epithelium
produce- a toxin (vboltragpri, cholera ertKfOtOxin,
cholera toxin, CT, or C T X) which is very similar
to the heat labile toxin (LT) of E . coif in structural,
chemical, biological and antigenic properties,
though CT is far more potent than I H in biological
activity. C T production is determined hy a
filamentous phage integrated with the bacterial
chromosome. It can also replicate as a plasmid
which can be transmitted ro nontoxigenie strains,
rendering theni toxigenic. C l , 1 CP and other
virulence factors arc regulated hy the T u R gene
product, ToxR pfotrin.
Polymyxin B ♦ "
sensitivity*
Group IV phage
susceptibility
Bl Tw phage 5 — +
niKCfftibilitt
* Strains isolated after 1^61 give variable results;
t 50 i. u. disc.
Ogaw.i AB
Inabl AC
Hiko^inta ABC
The toxin molecule, of approximately S4,000
M W consists of one A and 5 H subunits. The R
(binding} units attach to the GM. ganglioside
receptors on the surface of'jejunal epithelial cells.
The A (active) subunit, on being transported into
the entemeyte dissociates into two fragments A 1
and A .■ iTe A fragment onlv links the biologically
active A, to the B subunit.The A t fragment causes
prolonged activation of cellular adenylate delate
and accumulation of cAMP, leading to out pouring
into the small intestinal lumen, of large quantities
of warier and electrolytes and the consequent watery
diarrhea. The fluid secreted is isotonic with plasma
but contains much more of potassium and
bicarbonate. The toxin also inhibits intestinal
absorption of sodium and eh Coride. All clinical
manifestations and complications in cholera remit
from the massive water and electrolyte depletion
thu-. caused.
CT also exhibits other biological effects which
car be used for its detection a n d estimation. These

C o p y rig h te d m aterial
include activation ofllpolynls in rat tcstimkr tissue,
elongation of Chinese hamster ovary (CHO) cells
in culture and histological changes in adrenal
tumour (Y () cell culture and. Veru cells. Jt also
increases skin capillary permeability, and so has been
called the 'permeability factor’ (PF), It can be
demonstrated by the ‘skin blueing test' when CT is
injected rntradennilly in rabbits or guinea pigs and
pontamine shy bloc injected intravenously
afterwards, the site of toxin Injection becomes blue,
C T car also be estimated hy E L IS A . C l ts
antigenic and induces production of neutralising
antitoxins, C T can be toxoided.
Cholera vibrios also possess the
Upopolvsiccbaride O antigen ( l.l'S , endotoxin), ;ls
in Gram negative intestinal bacilli-This apparently
play? no role in the pathogenesis of cholera but is
responsible for the immunity induced by killed
vaccines. It may cause the fatal illness produced
experim entally by peritoneal inoculation in mice.
Cholera can occur in many
forms - sporadic, endemic, epidemic or pandemic,
India, mute specifically the large deltaic area of
the Ganges and Brahmaputra in Bengal, is its
homeland, where it has been known from very
ancient times. Till early in the nineteenth century,
cholera was virtually confined to India, periodically
causing large epidemics in different parts of the
country.
From 1617 to 192.1 cholera vibrios bad spread
from Bengal, in six separate pandemic Wives,
involving most parts of the world. It was largely
due to the threat of pandemic cholera chic
international health organisations catnc into being,
After the end of llie bih pandemic in 1923, till
1961 tlie disease remained confined to its endemic
areas, except for an isolated epidemic in Egypt in
1947,
The seventh pandemic originated from Sulawesi
(Celetas), Indonesia, in 1961 when the El 'lor
vibrios which hid been smouldering there to/ many
decades suddenly became more vnulcnr. After
spreading lo Hongkong .ind the Philippines, it
spread til caddy westwards, invading India in 1964.
By 19ffoT it bad spread throughout the Indian
subcontinent nird West Ariii- In the 1970s the
pandemic extended to Africa and parts- ■d Southern
Europe,
During; the course of the pandemic, the vibrios
had invaded affluent countries also. In the 1970s
small outbreaks had occurred in Queensland,
Australia and the Gulf Coast in the USA from
special environmental foci in die coastal waters.
However, they remained localised and were soon
controlled, in con Lost to the outbreaks in the poor
nation a, which developed into prolonged and
extensive epidemics.
In ramiary, 1991, the pandemic reached Hem,
thus encircling the globe in rhirty years time (Fig
33 2). fV-r the first time in the century, cholera had
invaded South America. 1 he epidemic spread
rapidly through many Central and South American
countries, and hy mid’ 1992 over halt a million eases
and 5000 deaths had been reported. By 1994 most
parts of Central and South America had been
involved and rendered endemic.
The Seventh pandemic of cholera has been
different from all the others, it is the first to have
originated from outside the Indian subcontinent, Ir
is also the first to have been caused hy the El Tor
biotype, in contrast to :J1 the earlier ones caused by
the classical choicra vibrios. The severity of illness
was much less, wit!] .l large proportion of r.uld and
asymptomatic infections. Mortality was low and the
carrier rate high, El Tor vibrios tended to remain
Endemic in many new geographic areas, causing
periodic epidemic bursts. The El 'lor vibrio baa
proved to be much hardier than the classical vibrios,
capable of surviving In the environment much
longer. A peculiar phenomenon has been the
replacement nt the classical bin-type hy the F,l Tor
vibrios following the pandemic spread. Thus, in
India the classical vibrio is hardly ever encountered
after the El Tor epidemic took root, though in
Bangladesh, the classical vibrio had staged a
comeback.
C o p y rig h te d m aterial
 An event of great Significance w an th e s u d d e n
emergence of non-Ql V! choletae (former NAG
vibrio) as the cause of epidemic cholera, In October
1992, i new non-01 vibrio was isolated from a
c h o le r a o u t b r e a k in M a d r a s ( C h e n n a i) . S im ila r
outbreaks soon fo llo w e d in d iff e r e n t p a rts of In d ia .
By January 1993, the new strain had become
e p id e m ic in B a n g la d e s h also. In [he affected arels,
this strain replaced the E l "lor vibrios as the
epidemic and environmental serovar. ic also showed
a tendency to be more Invasive, causing bacteiemic
illness in s o m e . The n e w epidemic s t r a in w a s
designated Serovar 0 - 1 3 9 (or 0 - 1 3 9 Bengal).
Unlike the 0 - 1 cholera vibrio, the 0 -1 3 9 vibrio is
capsulated. As it possessed novel surface antigens,
the 0 - 1 attain vaccines could not protect against
0 -1 3 9 infection. There was no natural antibody
against the scram in any human population then. It
wa$ therefore considered li kely that che O -139 strain
may initiate the nevt pandemic of cholera, The nvw
strain continued spreading, eastwards to tile South
Fast Asian countries, and westwards to Pakistan,
China and some parts of Europe, But surprisingly,
bv 1994 the El Tor Strain regained its .iimnn.im e
and the threat of an 0-1139 pandemic diminished.
Both O -1 t l Tor and 0 -1 3 9 srrui ns began i<tcoexist
in endemic areas.
Cholera is an exclusively human disease.
Infection originates from the patient or the carrier:
Carriers may he incubatory, convalescent, healthy
or chronic. Incubatory carriers shed vibrios only
during the brief incubarion period of 1 S days.
Convalescents may cxcieic them ftr l - . l weeks. The
healthy or contact carrier who ha?; had subclinieal
infection usually sheds [III1 vibrios lor 1c-.h than 10
days-The chronic carrier oOfth iuc- to ho active tor
months or years— the longest duration recorded
being 10 years. Chronic curriers were rare in

1994

E x p lo s iv e initial e p id e m ic s

E x ie n t of p a n d e m ic sp read

Unique environmental reservoirs

C o p y rig h te d m aterial
classical cholera but with E] Tor infection they sue .As cholera vibrios may die in a few hours at
seep more often. Persistent infection of the gall tropical temperatures, it is necessary to preserve
bladder Accounts for chronic carriage. the specimen at 4 °C or in some appropriate
Infection is acquired ch rough fecal) y holding medium. Stool samples may be preserved
contaminated water 0 1 food. Direct person-to- in VR fluid or Cuy-Blair medium for long periods.
person spread by contact may not be common but If the specimen can reach the laboratory in a few
hand contamination of stored drinking water has hours, it may be transported in enrichment media
been shown to be an important method of domestic such as alkaline peptone water or Monsur's
spread of infection. Large scale movement of medium, thus saving the time required for isolation.
persons, as occurs during fairs and festivals, has If transport media arc not available, strips of
traditionally been associated with the spread of blotting paper may be soaked in the watery stool
cholera. and sene to the laboratory packed in plastic
The persistence of the vibrio during the envelopes. Whenever possible, specimens should
intenepidemic periods was a matter of controversy be plated at the bedside and the inoculated plates
Jn the endemic areas it may be maintained by sent to the laboratory.
continuous transmission of su be bn Leal or mild Diagnosis by direct microscopic examination of
infection. It is now known that the natural habitat cholera stool is not recommended as the results
of cholera vibrios is the saline waters of coastal are not reliable- For rapid diagnosis, the
seas and brackish estuaries, where they can persist
for long periods r particularly in association with
small crustaceans such as copepods, crabs or
plankton.
A significant difference in susceptibility to cholera
has been reported in relation to blood groups, group
O percents being the most susceptible and group AB
the least- The reason for this in not known.
Stool, collected in the
acute stage of the disease, before [he administration
of antibiotics, is the most useful specimen for
laboratory diagnosis. Isolation of cholera vibrios
from such stools is a simple matter as they are
present in very large numbers, lO^1—10wvibrios per
mh The specimen is best collected by introducing
into the rectum a lubricated catheter and letting
the liquid stool flow directly into a screwcapped
container, Rectal swabs maybe used, provided they
are made with good quality cotton wool, absorbing
about 0 .1 - 0 .2 ml of fluid. They arc useful in
collecting specimens from convalescents who rtts
longer have watery diarrhea. In such cases, the swabs
should be moistened with transport medium before
sampling. Collection of stools from pans is not
recommended. VbmitUS i£ tIOt useful,

C o p y rig h te d m aterial
 314 * Textbook ol Microbiology *
character!-4tcmovllcy of the vibrio and its inhibition
by antiserum can be demonstrated under the dark
field or phase contrast microscope, using cholera
Stool from acute cases, or more reliably after
enrichment for six hours. Demonstration of vibrios
in htools by direct im[iiunolluoresCellce has been
attempted liut nonspecific fluowscence is common iuid,
the technique is too complicated for use in tire lieid,
On arrival in the laboratory; the specimens sent
in enrichment media should be i-icubatcd for 6 -8
hours including traits f time. The specimens sent
in holding media should be inoculated into
enrichment media* to be incubated for 6-8 hours
before being streaked on a selective and. a
nonselcct: l-c meditiim. It is also dc-Table to do direct
plating before enrichment The piling med-i used
vary in different laboratories hnt the media
employed usually are lule salt agar, MacCtionkey
agar for nunselecuve and TCBS agar for selective
plates. The plates should not be older than 3 -5
days and should be dried well befoit streaking. El
is possible to identify vibrio colonies on nonselectivc
media after incuhai n>n for 4-5 hours by exam [nation
under a stereoscope with obli que : Ilium: nation.
Generally the plates arc examined after overnight
incubation at 37^L\ Colonics suggestive o fvibrios
should be picked u, ith a straight wire and tested by
slide agglutination n-rh cholera O subgroup J
scrum (cholera 'nondiflcrenriarseium). If positive,
agglutmaNon may be repeated using specific Ogawa
and 1nabu sera for sen .-up ing. Hikqj ini a r,lins will
agglutinate equally well w:th Ogawa and Inaba. sera.
If agglutination is negative ^ ith nuc colony, if is
essential to repeat the test wirh at least five more
colonies* as 'agglui iuablc and DtMt-01 vibrlus may
coexist m the same specimen. 1: slide agglutination
is positive* the isolate is tested fur chick red cell
agglutination, This is employed for presumptive
differentiation between El Tor and classical cholera
vibrios. A report cat] be sent at ibis stage* usually
the day afrcr the specimen is received, [f no vibrios
are isolated* a second cycle of enrichment and
plating may succeed in some cases.
The isolate may then be subjected to detailed
study* if desired, including the oxidase test* ntilisari on
of aminoacids, lysine, arginine and ornirhine,
fermentation (]f sugar, including suernse, maurtoSe
and. arahinoRC, hemolysii* VP, polymyxin B
sensitivity and susceptibility ro cholera phage IV,
The strain may be sent to the International
Reterence Centre for vibrio ptliLge typing 41 the
National Institute of Cholera and Enteric Disease
(MICED) at Kolkaht.
I-■>!.=.!■.■- of vibrios that are not agglutinated hy
the O subgTmLp 1 scrum should not be ignored as
non -01 vibrios arc known to produce cholera-like
disease. An antiserum to the H antigen which :s
shared by all cholera virios has been found to be a
useful reagent. Any vibiio which is agglui maced
by th is H anti, crum* hut not hy O-T scrum is
considered to he nnn-Ol cholera vihrit]. Specific
antiiccum against 0 -1 3 ? is available. In the fully
equipped laboratory* diagnostic tests in cholera and
other diarrheal diseases should consist of a battery
of tesrs designed ro isolate other known pathogens
also.
Fur isul iliuu '.'I vibrios frrjsn Carriers*essentiallv
the nine techniques arc to be followed, except that
more than one cycle of enrichment may be
necessarv. As vibrio excretion is intermittent*
repeated stool examination will y:dd better results,
Examination of stools after a purgative (magnesium
sulphate 15—30 g or Mannitol 30 ,")* or of bile
after duodenal intubation is of special value.
Serological examination is of little use in the
d- tgnosis of cases though :t may be helpful in
asses ng the prevalence of cholera in an area. 'Die
tests av:1dable are agglutination using live or killed
vibrio suspensions, indirect hemagglutination,
\iIt i-.:, ii|.11 te.sr and antitoxin assay. Of these, the
complement dependent vibriocida] an ri body test is
the unosf useful.
For examination of water samples for vibrios*
c lit' ch ment or filtrat!<>n methods may be employed.
In the former, 900 m! of water are added to 100 ml
tenfold concentrated peptone water at pH 9.2,
C o p y rig h te d m aterial
 * V brio »
incubated at 37 for 6 -8 hours and a second
enrichment done before plating on selective media-
For the filtration technique the water to be tested
should, be filtered through the Mi 11ipore membrane
filter,, which is then placed directly on rhe surface
of a selective medium and meubared. Colo nice
appear after overnight incubation. Sewage should
be diluted in saline, filtered through gauze and
treated as for water.
Im m u n ity : Jn cholera, the vibrios remain
confined to the intestine, where they multiply and
elaborate the enlerutoxii] which is responsible for
the disease. Immunity therefore, may be directed
against the bacterium or against the rosin—
anrihaerenal or antitoxic. Nanr.il infection confers
some amount of immunity but it docs not seem to
last for more than /ml 2 months and reinfections
are lot-own after this period.
1mmum salient wi th killed vaccines 11 iduccs only
antibacterial immunity. The protective effect of these
vaccines, especially purified somatic antigens used
a* vull men, chough shori-lived, proves that
antibacterial immunity can protect agamsi infecti-nn.
The protection appears to be serotype specific hut
ricu biotype specific.
Immunity may be local, in the intestine, or
SvstCTnie. The appearance of local antibodies in [he
■nrestinc bas been known for a long rime. These
are known as ‘coproanti bodies' as they appear in
the feces. They consist of IgG, IgM and IgA.
Prophylaxis; The prevention of cholera requires
essentially general measures such as provision of
prureeted water supply and improvement of
environmental sanitation- As these ary noT easily
attainable, vaccination continues to be the most
widely used method ofprcvcnuon in endemic areas.
Cholera vaccines were introduced by Ferrari
within a year of the discovery of the vibrio. The
origin id vaccines were live suspensions of vibrios-
As chey gave n-:c to advene reactions, they were
replaced by killed vaccines. The vaccines used
traditionally are killed suspensions containing 3t)00
million V, cholera c pet ml, composed of equal
315
numbers of Ogawa and I tuba serotypes, given hv
suhe-utaneous or intramuscular injection. Many
laboratftri;:? employ clas-, ical cholera and El Tor
vibrio* in equal numbers in rhe vara inc. Strain
0 - 1 3 9 vaccine has also been prepared. The
concentriiri' in of the vaccine has been increased l -
0 ,0 0 0 million per ml, in -order to improve the
anrigerii stimulus.
Several con [rolled field trial's in endemic areas
with various types ol imccted vaccines have shown
that the protCCnon afforded h-v them di^'s notexceed
50-GG per cent; the durnrion of protection is only
,3—b months; the rate of protection m endemic areas
increases with age' a single dose of vaccine is
ineffective in children below five vears of age while
two doses at 1-4 week intervals are protective; a
-ingle dose confers good protection in adults due
to its acting as a booster on top of' prior natural
immunisation; ce ll-free somatic antigen
preparaliuns are as effective us whole cell vaccine!
then.- is good cross-protection between classical and
El Tor vibrios; the oobs- protection between Ogawa
and Inaba serotypes is doubtful and requires further
study, pe ruling which VJCcine Containing the
homologous serotype is to be employed. AJun linium
hydroKide and phosphate adi uva nt vac. i ne s
produced better immunity, particularly in young
children. Toxoid vaccines have not been been
successful. Injectable vaccines do not provide any
local immunity in the intestinal mucosa. They are
also unacceptably reaefogenit. I Hence attention has
been directed to oral vaccines.
Two types of oral vaccines have been Tried
recently, killed ora/ whole- ceil vaccines with and
without the inclusion of the B subunit ofCT, and
J)tf ora/ vaccines with classical, El lor and 0-1 3 ^
Strains, with their tmin genes deleted. While the
results have been pronii-irg, problems remain [O
be solved before they ate cleared far general use.
An ideal cholera vaccine is ycr to be found.
C holcra vatic inatum was a compulsi;rv requ irement
for international travel, but now very few countries
insist on elm.
C o p y rig h te d m aterial
316 * TexIO ook o f M ia o b io lo g y »
Treat me M: The treatment of cholera consists
essentially of the prompt anti adequate replacement
of lost fluid and electrolytes. Oral administration
of fluid containing glucose and electrolytes, cither
alone or supplemented by intravenous flu'.-.is is a
highly successful and freely available method of
treating chclera. Cereal hafied preparations ire
equally effective and usually more acceptable.
AntihacEen.il therapy i:- of secondary importance.
Oral tetracycline was recommended for redu.i,ng
the period of vibrio excretion and the need for
parenteral fluids. Inn illly cholera vibrio* were
unilormly susceptible to all antibiotics active against
Gram negative bacilli, but since 1979, multiple
drug resistant strains have become IncreisingEy
common.
VIBRIO MIMICUS
So named because it closely resembles cholera
vil>rios in bioeliemical fearures, V, lahaiais can be
di lFeren li.Lied by its failure to ferment sucrose. Like
V, cholenc, it grows best at low sale concentrations
(0..5-l%). Tt has been responsible for many spnr&H:ic
cases of diarrheal disease on rhe Gulf Coast of the
USA, Infection is acquired from eating seafood,
especially oysters. [ :ic di-easc is seif--incited.
Clinical manifestations resemble those caused by
V, parahacniolvticus.
HALOPHIUC VIBRIOS
Vibrios that have a high requirement of sodium
chloride arc known as halophilic vibrios. Their
natural habitat is sea water and marine life. Some
hilophiilC vlba iO$ have been shown tu cause human
disease— K /wraJutejiiaJKfjLUJi, V. algiimiyncus and
V. vulnificus.
V ib r io P a r a h \e m o l i t ic u s
V. pitah&CinolytiLus is an enteropathogcn'ic
halophilic vibrio originally isolated in 1951 in Japan
as the Causative agent of an outbreak of food
poisoning due tu sea fish- Gastrocntcrfis due to
this vibrio has since been identified in several
countries and it is now considered an importaot
cause of food poisoning throughout the world. It
inhabits rhe coastal seas, where ir is found in fishes,
arthropods such as shrimps and crahs, and molluscs
such as oysters. In Calcutta, it has also been found
in small pond fishes.
In morphology, it resembles the cholera
vibrio, except that ir is capsuiated, shows bipolar
staining and has a tendency to pleomorphism,
cspc-inlly when grown on 3% salt agar and in old
cultures. Unlike other vibrios, it produces
peritricboust flagella when grown on solid
media. Molar flagella arc formed in liquid
cultures.
ir grows only in media containing NaCl. It can
tolerate salt concentration upto 8 per cent but not
10 per cent. The optimum salt concentration is 2­
4 per cent. On 1 C h S agar, the colonies are given
with an opaque, raised centre and flat translucent
periphery. The string test is positive.
It is oxidase, catalase, nitrate, indole and citrate
positive. Glucufie, maltose, mannitol, mannose and
arab'inose are fermented producing acid only.
Lactose, sucrose, sal kin, xylose, adonltoi, Inositol
And sorbitol are hoe fermented.
It is killed at b0 aC in 1,5 minutes. It docs not
grow at 4 "C but can survive refrigeration and
freezing. Drying destroys ie. It dins in distilled water
at vinegar in a few minutes.
Three antigenic components have been
recognised— soma lie O, capsular K and flagellar
H antigens. Scrotyping is based on O and K
antigens; \2 O groups have been recognised and
59 di-dnet k antigens.
Not all strains of Vr parahaemolyticus are
pathogenic for human beings, It has been found
that strains isolated from environmental sources
(such as water, fish, crabs or oysters} are nearly
always nonhemolytic when grown on a special high
salt blood agar {Wagatsuma agar), whi'e strains
from human pnvL-nfs are almost always hemolytic,
'fbii-. is called the Kanagawa phenomenon and is
due to a hear stable hemolysin. The significance of
C o p y rig h te d m aterial
 Inflate * t
V.P
Migrate reduction ■+
[Jreue —
Sucrottfermencatioh
Swarming
Growth in CW NaCl
7% NaCl + *
i m NaCI
thin htiruilysis i:- not known hut it in u-sed an a
laboratory test for pathogen i-cLtjr; Kanagawa positive
strains he jpg considered pathogenic tor human
beings. and negative strains nonpathogcnic. Nn
enterotoxin has been identified- The vibrio is
believed to cause enteritis by invasion of [he
intestinal epithelium. V, purdhacmolyTkiss causes
food poi son ing assoc iated wi th man nc food.. 1r also
causes acute diarrhea, unissociatcd with food
poisoning. Abdominal pain, diarrhea, vomiting and
fever arc the usual signs, Feces contains cellular
exudate and often also blood. Dehydration is of
moderate degree and recovery occurs in 1-3 days.
Cases arc more common in summer, aod in adults
than in children. In Calcutta, V. parahaanolyticus
could be isolated from 5 - 10 per cent of diarrhea
cases Admitted ro the Infectious Diseases Hospital.
V. p&rzhiemohncus is common in sea fish in some
other parts of India but human cases are much less
frequent.
This halophilic vibrio resembles K p&fihaeiilO-
JVfrfUS in many respects and was tormcrlv considered
Abiotype of the Utter, It has a higher sail tolerance,
is VT positive and ferments sucrose ^Table 33-6),
It is frequently found in sea fish. Its status as a
human pathogen is uncertain. It has been associated
wi th infections of eyes, cars and wounds in human
beings exposed to sea water.
t
*■
+
+
*■
V vulnificus, previously known as L* vibrio or
iJenedtea iriltufica, is a marine vibrio of medical
im|ujrtance. It is VP negative and ferments lactose
but not sucrose. It has a salt tolerance of less than
eight per cent. It causes two Types of illness. The
first is wound infection following contact of open
wounds with seawater. The second rype occurs in
compromised hosts particularly those with liver
disease. Following ingestion of the vibrio, usually
in oysters, it penetrates the gut mucosa without
causing gaxttoin tei final manifest it ion's and enters
the bloodstream, rapidly leading to septicemia with
high mortality.
Besides the genu* VJfirio, rhe family Vibrionaceae
,l1eo contains the genera Aerom onas and
Ple.siojrtt/jTas, some members of which have been
associated with human lesions.
Aemmonas hydropAjJa, originally isolated from
frogs, in which it causes the Lred icy disease', has
been reported from many cases of diarrhea and from
some pyogenic Lesion* in human beings.
PitsiomoDis shigelloides also has been reported
from diarrheal disease. Both these ire oxidase
positive, polar flagellated, Gram negative tods Aild
may be mistaken for vibrios. They may be
differentiated froin vibrios by biochemical test* suc-h
a* utilisation of aminoacids.
C o p y rig h te d m aterial
 * TexlDoo* ol WicrabiolDBV +

fu rth e r Reading
Rama D iiiiii WR CieuJKiLigJi 1992. C h o lea . New YoriiPlenum.
Collier L ec al leds). 199U. Toptcv m d WiTJi.imr M icrobiology in d M k ro b iil Inteci^mSy 9*1, edn. London ; Arnold Vala. 2
and J.
C tih n lf flR jrid A H m ] 1994. fjM'Jeoonjenraj1r« w -: nr of Vibrio ch a tem . .-l.-Jn.L.1- A c ,<■■.i Sci 740:44.
hir.:.| 5M Cl lli KcrlEcgccLji.'c acid £Ytkluli^rt ol Vr. ■.11■:-1 i llu 0 -1 3 9 . Pr&i N it A cad S^i i.USA} 100; 1304.
Honda T undT |5da 1.99,1. Tile pirliLt^tnidw of ViiwiN) p u rth icm o ln ictis. &b'McrfjVficn>bt0£ 4:lGfi.
Kaper JEi cr ill. 1995. Cholera. C'Jin Miiyob'i-j) fteiv SsH.
l.L. eySW. 1995. (. ::nLert. C lin I n f e r D is. 20tl4O9.
71.1:■i:.l! .i-: : ■■- r|and JC >.i.I-: 11 1994. Cliolrhi vaccines. ! \; r; : i c 245:OS7.
N aifG Bcial. 1996. Vibrio e/io^raeOl39'Bengal. Jfo FAferGVfjicra(jioJ7:43.

C o p y rig h te d m aterial
 Pseudomonas

Pseudomonas ire a large group uf aerobic, on Mactonicey and DCA media, forming non­
nonsporing Crum negative bacilli, motile by polar lactose-fermenting colonics. Many strains ate
flagella. They are ubiquitous, mosdy saprophytic, hemolytic on blood agar. In broth, it forms a dense
being found in water, soil or other moist turbidity with a surface pellicle.
environments. Some of them art pathogenic to Ps. aeruginosa produces a number (if pigments,
plants, insects and reptiles. A tew cause human the best known being pyocyanin and fluorcscin.
infection, typically opportunistic- Pyocyanin ^ a bluish green phenamine pigment
Based on moleeularanalysis, pseudomonads have soluble in water and chloroform. Fluorescin
been reclassified and many former Pseudomonas (pyoverdin) is a greenish yellow pigment soluble
species reallocated to new genera such as in water but not in chloroform. In old cultures it
VJurk/io/derid, StenoCTophomonas and others. may be oxidised to a yellowish brown pigment.
Pyocyanin is produced only by Ps. aeruginosa but
fluorcscin may be produced bv many other species
Ps- p y ocy a n c a; B a c i f f a s p y ocy sn cu s also. Other pigments produced are pyorubin [red}
It is a slend er C ram negative and pyomelanin (brown) in various combinations.
bacillus, 1.5—3 pm * 0 .S pm, actively motile by ™ $r>mestrains maybe nonpigmented- It is not known
polar flagellum. Occasional strains have two or whether the pigments have any role in
three flagella. Clinical isolates arc often pillared, h pathogenesis. Some of the pigments particularly
is noncapsulated but many strains have a mucoid pyocyanin, inhibit the growth of many other
dime layer. Mucoid strains, particularly isolates bacteria and may therefore contribute to Ps.
from cystic fibrosis patients have an abundance of aeruginosa emerging as the dominant bacterium Lo
extracellular polysaccharides composed of alginate mixed injections..
polymers. This forms a loose capsule (giyeocalyx) The metabolism is
in which microcolonies of the bacillus are oxidative and nonfermentative. Peptone water sugars
enmeshed and protected from host defences. are unsuitable for detecting acid production, since
It is an obligate this is weak and gets neutralised by alkali produced
aerobe., but can grow anaerobically if nitrate is from peptone. An ammonium salts medium in
available- Growth occurs at a wide range of which the sugar is the only carbon source is the
temperatures, "C, the optimum being d7 “C. best. Glucose is utilised oxidatively, forming acid
It grows well on ordinary media, producing large, only Indole, MR, VP and H ,S tests are negative.
opaque, irregular colonies, with a distinctive, musty, Nitrates are reduced to nitrites and further to
mawkish or earthy smell. Iridescent patches with gaseous nitrogen. Catalase, oxidase and arginine
a metallic sheen are seen m cultures on nutrient dihydrdast tests are positive,
agar. Crystals are seen beneath the patches. It grows As f t . aeruginosa has become

C o p y rig h te d m aterial
m i Textbook of fc^icFobtotofly ►

a very important cause of hospual L]itulTkjils, US community outside the hospital, the most common
classification i-i essential for epidemiological infection caused by f t . aeruginosit is suppurative
purposes. Serocyping, bacreriocin (pyocin, . otitis, which is chronic though not disabling. In
acruginoHin) typing and bacteTiaph.ige typmg have the hospital, it may cause localised or generalised
been used but are not entirely satisfactory. infections. L-nca Used lesiOM ire cnrnmnnly
Restriction endonuclease typing with pulsed-freld infections of wounds and bedsores, eye infections
gel electrophoresis is the mast reliable method and urinary infections following catheterisation. Fs.
available aeruginosa is the most common and most serious
Resistance: The bacillus is not particularly heat cause of infection in bums. It l? also one of the
resistant* being killed at 55 nC ir: one hour but agents responsible for iatrogenic meningitis
exhibits a Irigh degree of resistance to chemical following lumbar puncture. It frequently causes
agents. It is resistant to the common antiseptics and post-tracheostomy pulmonary infection. Septicemia
disinfectant* such as quaternary ammonium and endocarditis may occur in patients who arc
compounds, chloroxylenol and hexachlorcph&nc debilitated due to concomitant ielection, malignancy
and may even grow profusely in bottles of such or immunosuppressive therapy. Ecthyma gangreno­
antiseptic lotions kept for use in hospitals- Indeed, sum and many other types of skin lesions have
selective media hive been devised lor JV. aeruginosa beer described occurring cither alone or as
incorporating dettol or cetrimide, It is sensitive to part of generalised infection, mainly in patients
acids, beta ghiraraldehyde, silver salts and strong with leukemia and other types of malignancy.
phenolic Ji sirliettants. Its suacept ibibty to si her has Infection of the nail bed i.s not uncommon
been applied clinically in the use of silver following excessive exposure of hands to detergents
sulphonamide compounds as topical cream in and water.
bairns. 1\ iertiginuSa his been described as one of the
Psr aeruginosa possesses a considerable degree agents responsible for infantile dianfrea and sepsis.
of natural resistance to antibiotics. Examples of Strains isolated from outbreaks of diarrhea may
cLinicaJly effective antibiotics are aminoglycosides form a heat labile cnteroloxin and give a positive
(gentamicin, amikacin), cephalosporins (cefotaxime, rabbit ileal loop reaction. F.-. aeruginosa has been
ceftazidime, cefoperazone), fluoroquinolones reported to cause a self-limited febrile illness
(ciprofloxacin, ofloxacin, pcflnxacin), penicillins (Shanghai ifcveft resembling typhoid fever in some
(piperacillin, ticarcillin, azlocillin). For localised tropical areas.
infetums, topical colistin, polymyxin B or 1% acetic The pre-eminent role of Ps. aeruginosa in
acid may he useful. hospital infection is due to its resistance to common
P n lh o g e n r c tt}: "Blue pus1 was known as a antibiotics and antiseptics, and its ability to establish
surges] enriiy longhefone Gessvd (1SS3) isolated itself widely in hospitals. Being an extremely
f t . aeruginosa from such cases. Both the specific adaptable organism it can survive and multiply even
names of the bacillus refer to its capacity to cause with minimal nutrients, if moisture is available.
“blue paifiFTthe term aeruginosa, meaning verdigris Equipment such as respirators and endoscopes,
which is bluish green in colour and pyocyana, articles such as bed pans and medicines such as
being a literal translation of 'blue pus1. lotions, oi iLiments and eye drops and even stocks of
The pathogenic importance of the bacillus was distilled water or plants and flowers may be
not adequately recognised till recendy, when it has frequently contaminated- P& MTUginaM is present
established itself as one of the most troublesome on the skin of the axilla and perineum in some
agents causing nosocomial infections, in the persons. Fecal carriage is not common but may he

C o p y rig h te d m aterial
frequent following oral antibiotic treatment or
hospitalisation.
The mechanisms of pathogenesis :tn: not
clearly understood. Several toxic extracellular
products have heen identified in rhe culture
filtrates, such as ewotoxins A and $, Eflotewin
A acts as in N A D ise, resembling the
diphtheria toxin. Good antibody response to
CKOtoxin A is considered a favourable sign in
severe infections with Pi. aeruginosa, Other
toxic products include proteases, elastises,
hemolysins and entenitentin. The slime layer
acts as a capsule in enhancing virulence.
The bacterium grows
readily on moat media.The identification of
pigmented strains of the bacillus from clinical
specimens is easy. Bui about 10 per cent of isolates
may be nonpigmented. Prompt oxidase reaction and
arginine hydrolysis help in their identification, It
may be necessarv to use selective media -such as
cetrimide agar for isolation from feces or other
samples with mixed flora. Aa Pa. j efuginuaz is a
frequent contaminant, isolation of the bacillus from
a specimen should not always be taken as proof of
its etiological role. Repeated isolations help to
confirm the diagnosis.
Prevcntion of Ps- aeruginosa cross-
infection in hospitals requires constant vigilance
and strict attention to asepsis. Antibiotic treatment
is not always ■satisfactory. Animals with experi­
mentally infected bums have been protected by prior
immunisation with the homologous strains.
Immunotherapy in human burns Cases with
antiserum, to Ps, aeruginosa may be useful-
Pseudomonas vaccines are being tried in CyStLC
fibrosis patients who arc highly vulnerable to
pseudomonas infection.
Specific antibacterial therapy constitutes only
one aspect of the management of serious
pseudomonas infections. Treatment of the
underlying diseases, correction of granulopenia and
appropriate supportive therapy need attention.
Occasional opportunist infections may be
caused hy a few other species* such As Ps.
fluorescein, Fs. puiida and Ps. stutzch.
Tbit; is a saprophyte and opportunistic pathogen,
causing wound infecticm, uri nsuy tract infectio n a nd
septicemia. It is usually oxidase negative and
acidifies maltose in addition to glucose, lactose and
sucrose. Infections usually respond to cotrimoxazole
and chloramphenicol.
This is a plant pathogen causing onion rot (cepia,
Latin for onion). It is increasingly bci ng recognised
as. art Opportunist environ inert Cal pathogen,
particularly in those wirh cystic fibrosis or chronic
granulomatous disease, in whom it causes fatal
necrotising pneumonia, It is nutritionally very
versatile. It can grow in many common disinfectants
and can ever use penicillin G as a sole source of
carbon! It Is oxidase positive and acidifies mannitol,
sorbitol and sucrose. It can cause urinary, respiratory
and wound infections, peritonitis, endocarditis and
septicemia. It is inherently resistant to most
antibiotics.
Tire bacillus had also been classified variously
as L o cftlcrclh , P fciffcrcJh , M alhom yccs.
Actinubacillu* and Acmetohlfter It IS the
causative agent of glanders (raai/tuv. in Latin}, a
disease primarily of equine animals - horses,
mules and asses - but capable of being
transmitted to other animals and to human
beings, r|Tie bacillus was discovered by Locffler
and Schutz (1882).
Ps. matter is a slender, nonmotile, Gram negative
bacillus, 2—5 pm * 0.5 pm staining irregularly and
C o p y rig h te d m aterial
often giving a beaded appearance. Il is in aerobe
and facultative anaerobe, growing on ordinary media
under a wide range of temperature. C ulonici which
are small and cranslusecrt initialiy become yellowish
anil opaque on Lining, On potato, a characteristic
am her, honey-like growth tp pc .If*, becoming
greenish mellow resembling Ps. acnjgimm, I l is
quite inactive biochemically attacking only glucose.
The natural disease in equine z occurs ■i'l two
forms— glanders and farcy. In glanders, the
respirators-system is involved, with the formatLOH
nf firm, round nodules and a profuse catarrhal
discharge from the nose. Farcy follows infection,
through the skin und as an involvement of the lymph
vessels and nodes, which stand out as hard cords
beneath the skin.
Guinea pig£ are susceptible and in iT a p e riro n c a l
injection into male guinea pigs induces the &TttlS
reaction. This oinsists o f swelling of the testes,
inflammation of ninica vaginalis and ulceration qf
the scrotal s k i n . The S traus r e a c t i o n is n o t
d ia g n o s tic of glanders, a* it may also be produced
by inoculation of other b a c te ria such as Brucella
species, Preisz-Nocard bacillus, Actina&sCxlhu
tignietti a n d P i. ffscudvitmlh-i.
\luman inteetion is usually occupational, found
in ostlers, grooms anti veterinarians. It mav be acute
or chronic and is pro re an in character, with
localisation in the respiratory tract, skin o r
subcutaneous tissues. In acute glanders, there is
fever, mucopurulent nasal discharge and severe
prostration. The fatality rate is high. While human
infection is acquired only rarely from infected
animals, Laboratory cultures are highly infectious
and Fs, mallei is one of the most dangerous bacteria
to work with
Animals suffering from glanders develop a
delayed hypersensitivity to the bacterial protein.
This is the basis of the mtUtifi rey[ used for
diagnosing glanders. This is analogous to the
tuberculin test and may be performed by the
subcutaneous, Sntracutancous or conjunctival
methods.
Also known as Whitman's bacillus, Actinobacillus
wfijfTncVT, jV/aMctvn rc-es pscudom^Jlci, LjxiBetelli
psvudomallct).
This is the causative agent of meliuidusii, a
glanders-liltc disease, epizootic in rodents in
southeast Asia, India and North Australia. (The
name is derived from jtfcJj.s, a disease of as-seS
[glanders], and cj'tfos meaning resemblance). The
disease was first described in human beings by
Whitmore and Krishnaswami (1912) in Rangoon.
Whitmore (1913) isolated the bacillus. It resembles
Ff, m i l l e l but differs in being motile, Liquefying
gelatin and forming acid from several sugars. Two
thcrmolabile exotoxins, one Lethal and the other
necrotizing have been identified in culture filtrates.
The human disease may take different forms. I f
[ray be an acute septicemia, a subacute typhoid-
hkc disease, or pneumonia and hemoptysis
resemhlingtulierLulosLS. In chronic form, there may
be multiple caseous or suppurative find, with abscess
formation in the skin and subcutaneous tissues,
bones and internal organs. Acute melioidosis has a
high case fatality rate. Serological evidence indicates
that inappaient infection is Common in endemic
areas. Long latency ami reactivation may occur an
the bacillus can survive int race Hula rlv in the
reticuloendothelial system. The bacillus ha£ been
isolated from water and soil in endemic areas. It is
a soil saprophyte that causes infection in rodents
and humans accidentally. Human infection occurs
commonly through skin abrasions or by
inhalation.
Diagnosis maybe made by demonstration of the
bacillus in exudates by microscopy Ismail irregularly
staining Gram negative bacilli, showing typical
bipolar 'safety pin’ appearance wirh methylene blue
stain), isolation bv culture from sputum, pus, blood
oi urine, or by serology {F1LISA for IgM and lgG
C o p y rig h te d m aterial
 < Pseudomonas * 323

antibody; indirect hemagglutination). A PCR teat cotrLmoxazole, tetracycline, amoxycillin davulanatc,

has also been developed- or chloramphenicol. Prolonged treatment, for many
Ceftazidime is the drug of choice, along with months maybe necessary.

Further Reeding
B iltc h A L and Sm ith R P (td s ). 1994. f^ru doFrw jiu i o i ( p i u M infections N fw Y o rk : M arcel Dekker.
D iner DA£. I W . MelinJuEis- L.".'in .Uirni.'Mui R - .r . 4^2,
Fick RB (cd), 1992. An.'i/itmu'M- flerw?im?H: 77te Prthogrnwif ant? Pise***. Rev* RatcmiCRC-
tj-lUgBn FR, 1995, Fseudomina* and Rurkhnlderi.i In Murray I’ R c( ai Afunual o f f.7mir.n' Afrcrrhiofe^y.
Wi'liLiiyliiii:AmL'iii':m o f M um Pi. Jugy
f I m i k: L M .m d K R i^ 1 1985. P se u d o m o n a s aeru g in o sa - C h r iia il m a n ife s ta tio n s and m a iH R e m e n r. L a n c e t 2:1224,
L w la ra s a m e e A and S. E m n w o n k itti 1 9 8 9 - M f llk iiilw is . R e v fnfrtf Dir- 11:413-
M o m m r A | Jr, a n d R P .W fe rK l 1984, li]n !d e m :.i> lo g y o f i n f e t f o n - d u e to P ieudo m cm as -ie ru £ in « a . Re-v in fat Da. d rS u p p l
5-627-
lJatama«JKon F ct al. 1982. M e lio id n s i* ./ f t d j> ir ] O il 175-

C o p y rig h te d m aterial
 Yersinia, Pasteurella, Francisella
The plague bacillus and many other Cram negative,
short bacit!] iliac an-! primary pathogens of rodents
were grouped together in the genus PasteureHz.
Based on cultural and biochemical differences, rhi>
giuup lias been divided into tluree genera - Versima,
Pastmrclta and Fr^ncis^ila. The genus Vcr.sjJTja,
containing the medically important species Yiptsris
(the causative agent of id ague), Y. ps'etiJo
tulmttdbili; {a primary pathogen of undents) and.
Y. GnteraootiticM (which causes enteric and systemic
diseases in animals anti human beings) was so
named al ter Alexandre Yersin, who discovered the
plague bacillus- The genu& Itrs/tnii is now assigned
to the family Enterobacteriaceae. The genus
Pasreurt?Ua contains several related bacteria causing
hemorrhagic septicemia in different species of
animals and occasionally producing local and
systemic infections in human beings, grouped under
a common species named E multod da. One of diese,
P avisepFfta is the chicken cholera bacillus used
by Pasteur for the development of the first attenuated
bacterial vaccine. Hence the name PhsteurelL. The
genus Francis.Hu, consisting of F. futarmsis, is
named after Francis for his pioneering studies on
tularemia, caused by this bacillus.
The plague bacillus was discovered independently
and simultaneous Iv hy Yersin ami Kita^ato (1S^4)
in Hong Kong at flic beginning of the List
pandemic of the disease,,
Y. peers' is a short, plump, ovoid,
Gram negative bacillus, about 1.5 pm * 0-7 pm in
sire, with rounded ends and convex sides, arranged
singly, in short chains ocin small groups, hi smear*
stained with tiiernsa or methylene blue, it shows
bipolar staining (safety pin appearance) with the
two ends densely stained and the central area dear
{Fig. 3 5.1). Plenmorphism is very common and in
old cultures, involution forms are seen— coccoid,
club-shaped, filamentous and giant forms.
Pleomorphisim is vh anicteric ally enhanced in media
containing 3% NaCL
The bacillus is surrounded by a slime layer
{envelope or capsule). Jt is nonmotile, nonspuring
and 11011 a< id fast.
The plague
bacillus is aerobic and facultatively Linaerobic.
Gmwth occurs over j whlc range of pi l [pi I 5“9.6,
opti mum p! 1 7- 2 ) and temperature (range 2~45 “CJ,
The optimum temperature for growth (unlike most
pathogens) ]S 2? ‘'C but the envelope develops best
at 37 (rQ-
1t is not nutritionally exacting and grows on
oidin.irv media. O r nutrient agar, colonies arc small,
delicate, transparent discs, becoming opatjue on
continued incubation. Colonic* on blood agar or
other hecniu containing media are dark brown due
to the absorption of the he mid pigment. Colourless
colonics arc formed on MacConkey's agar. In broth,
a flocmlent growth occurs at the bottom and along
the sides i>t the tube, with lutlc or no turbidity. A
delicate pellicle may form later, If grown in a flask
of broth with oil or ghee (clarified butter) doited
on top (ghee broth) a characteristic growth occur1,
which hangs down into the broth Irom the surface,
resembling stalactites {Fig. 35.2).
C o p y rig h te d m aterial
 i Glucose, imkosr and
mmnitol but not lactose* sucrose or rhamnosc arc
fermented with the product: rail of acid hut no gas.
Indole If- rtnt produced. It i* MR positive and VP
and citrate negative, catalase positive and acsculin
positive and nJodusc acid Itieut negative, Grlatm Is
not liquefied, Eased on tint fermentation of^ycetol
and reduction of]liIrate* Devignal has distinguished
threephysiological varieties of Vi/MSftj.s.This typing
appears to be of epidemiological significance
because of the different geograph lcj] distribution
of the types (Table 35,1),
' The plague bacillus is easily
destroyed by exposure to hem, sunlight, drying and
chemical disinfectants. It is destroyed hy heat at
.55 L'L’ or by 0.5% phenol in 15 mi notes. It remains
vi able for long pc ri ods in cold, moist envi ron mems,
It can survive for several months*arid even multiply,
in (lie soil ot rodent burrowv. AH strains arc lysed
by a specific antiplaguc bacteriophage at 22 “G.
/ jjS r J ' ■.i 'I" .' Cr . ' r' '■ ’i :
■ Plague bacilli arc antigenically
homogeneous and scrotypc-s do not exist. The
antigenic structure is complex. At least 20 antigens
have been detected by gel diffusion and biochemical
analysis, Many of them have been chimed to he
virulence factors. Tliev include the following:
1. A hear labile protein envelope antigen (Friction
l or F-I) Ih: sL feruled in cultures incubi ted at
37 nC- It inhibits phagocytosis and is generally
present only in virulent strains. This plasmid
encoded antigen has been considered a virulence
determinant but occatiooill strains deficient in
Fraction 1 antigen have been isolated trout fatal
human cases, The antibody to this antigen is
protective in mice.
2. Two antigens designated V and W and always
produced together have been considered to be
t he virulence (actors a s they inhihit phagnrytos is
and intracellular killing of the bacillus,
Fruduction of V and W antigens is plasmid
mediated.
C o p y rig h te d m aterial
d. Virulent strains [iroduce a fcHicieriocin {Pesth-in 1)*
coagukse and fibrinolysin, Pcsticin l inhibits
strains of Y. pMudorukejruVojfis, Y entero
colitica and E. coti.
4. The term 'plague toxins' refers to It least two
classes of toxins found ir culm re filtrates or
cell Lysates. The first is the endotoxin. a
lipopolysucchuriJe similar to the endotoxins of
enteric bacilli. The second class of toxins is
protein in nature t possessing some properties of
both exotaxins and endotoxins. They are
thcnnolabilc and may be traoided Hur do nor
diffuse freely into the medium and arc released
only by rhe lysis of iliti cell. They are called
murine ftnuH as they ,irc active in rats and mice
but nor in guinea pigs, rabbits and primates. On
injection into experimental animals, plague toxins-
produce local edema and necrosis with systemic
effects on the peripheral vascular system and liver.
The role of plague toxins in natural disease in
human beings is not known.
5. Virulence also appears to be associated with an
unidentified surface component which absorbs
heiniil and basic aromatic dyes in culture media
to torm coloured colonies.
Y. parts v ie orientals - *
Y. pettit rar. an Cjljli a + *■
' . pctlis nrr. Jiwdieva/jt + -
h. Virulence has also been associated with the ability
for purine synthesis.
Plague is ait ancient sCOurge of mankind. The
disease was familiar To the ancient civilisations of
Asia. The B/iagavar/ut P lticuj urged householders
to flee when rat falls were noticed.
Central Asia or the Himalayas is believed to
have been the original home of plague* from where
ir has, in wave after wave, spread fir and wide,
causing epidemics and pandemics, exacting a toll
of human life surpassing any other disease. The
identity -of the Rihlical plague of the Philistines
(1,120 Be) is in doubt hut the pandemic that
occurred during the reign of EmperorJustinian {AD
542] was undoubtedly bubonic plague (believed to
have been caused by Y, pestis, var. antj'rjud) and
caused a hundred million deaths. In the fourteenth
century, jtanderruc plague known as the ‘black death'
is hclieved to have kitted j quarter of all mankind,
(war. m cdicvalis believed responsible). The name
'black death’ mav have been derived from the
extensive cutaneous hemorrhages and gangrene
often seen in fatal cases of plague-
Primacy foci in India,
Myanmar; and China.
Causative agent of 1894
pandemic. Responsible for
wild plague in Western USA
South America, South Africa.
Transbaikalia, Mongolia,
Manchuria, perhaps
responsible for Justinian
plague.
S. utheast Russia.
C o p y rig h te d m aterial
 i Yortinla Pas‘ei/rnlla FraodSfllta ►
Historians of plague identify 41 epidemics
before thq birth of Christ and 109 epidemics in
ihe next 15 centuries. There are records of 45
pandemics between AD 1500 and 1720, The disease
was quiescent in the eighteenth and nineteenth
centuries and confined to endemic foci. The last
pandemic started in Hong Kong in lE94and spread
throughout tlie world, (caused by F,pearls van
orfen tails). India was one of the countries worst
hit by this pandcmii r Plague reached Bombay in
1H96 and spread all mver the country during the
next few years, causing more than 10 million deaths
by 191S. Ir gradually receded thereafter, though
occasional cases continued to occur in endemic foci
till 1967. No further plague cases were seen in India
rill 1994, when in August a nonfatal outbreak of
bubonic plague was reported from Maharashtra
(Seed district). In September pneumonic plague
was reported in Surat and ad: oining areas of Gumrar
and M,iharashir.i, causing much panic and
consternation. A few cases were reported from
different pans of north India also, probably caused
by the exodus from affected areas. During the
outbreak which subsided in two months, there were
over <i(KK> suspected plague cases and 60 deaths. In
February 1002, plague struck again causing a short
outbreak near Simla, claiming 4 lives.
Plague survives in several scattered natural foci
in many countries (Fig. 35.3) in wild rodents,
occasionally causing infection in human contacts.
In India at least four foci, of plague arc known. One
is the region near Kolar at the trijunction of Tamil
Nadu, Andhra and Karnataka. The second is the
Becd-Latur belt in Maharashtra from where the
Surat epidemic emanated- The third is in Rhoru in
Himachal Pradesh where the 2002 outbreak took
place* and the fourth is a small pocket in
t Ittaranchal.
In human beings, plague occurs in three major
forms: bubonic, pneumonic and septicemic. In
bubonic plague, after an incubation period of 2-5
days, the lymph nodes draining the site of entry of
the bacillus become Infected. In some* the infection
327
remains localised at ibe site of flea bite, with only
minor constitutional symptoms (pcstis minor). As
the plague bacillus usually enter* through flea bites
on the legs, the inguinal nodes are involved and
hence the name 'bubonic’ l.bubon meaning groin).
The gland* become enlarged and suppurate. The
bacilli enter the bloodstream and produce
septicemia- Sometimes there are hemorrhages into
the skin and mucosa. Disseminated intravascular
coagulaiion is cummun and may lead to gangrene
of the skill, finger* and penis. The case fatality in
untreated cases may be 30-90 pet cent.
Pneumonic plague may be seen sometimes
dun ng eji idemies of bubunit plague. Rarely, pr» inary
pneumonic plague may occur in epidemic form, as
happened in Manchuria during 1910-1912, causing
some 60,000 deaths. Pneumonic plague i> spread
bv droplet infection. The bacilli spread through
the lungs producing hemnrrhapLc pneumonia.
Cyanosis is very prominent. The bloody mucoid
sputum that is coughed out contains bacilli in
enormous numbers. Pneumonic plague is highly
irtfcctjous and in untreated patients, almost
invariably fatal.
Septicemic plague is usually the terminal event
in the hubonic or pneumonic plague but may
sometimes occur pri man ly. Mcningi tic involvrment
may occur rarely. Human carriers have not been
recorded hut asymptomatic oropharyngeal infection
has been observed in some contact*.
Epidemriold^y? Plague is a zoonotic disease.
The plague bacillus is naturally parasitic in rodents.
Infection is Transmitted among them by rat fleas.
The fleas acquire the infection by feeding on
infected rodents. In the flea, the bacilli mulriply in
the stomach to such an extent that they block the
pruventriculus. The interval between the ingestion
of infected blood and blocking in [he proveiltficulus
is the extrinsic incubation period, which is usually
about two weeks in XcnapsyUa cheopk. When such
a 'blocked flea1 fore* another rodent, it cannor suck
in blood because the bacterial mass blocks the
passage illn hanic*lly- The blood, mixed with the
C o p y rig h te d m aterial
 YERSINIA. 1hASTL L!'BELLA. LRANL'ISEL LA
bacteria is regurgitated into the hire, transmitting
the infection. Infection may also be transferred by
contamination ot tluc bite wound with the feces of
infected fleas. When a diseased rut dies (rat fall),
the .fleas leave the carcass and in the absence of
another rat, may hire human hidings, Causing
IhiIki iLie n Iague.
Sfwfa! species of fkas may act as vectors, the
most important being XtAOpsyil* chcopis, X u t a
and C-cratophyflus A l d a t w , X . ch co p i p, the
predominant species in north India is a more
efficient vector than the south Indian species A",
aifia. This has contributed to the more extensive
nature of plague outbreaks in the north as compared
to south India. Plague epidemics generally occur
in the coni, humid seasons that favour the
multiplication of fleas, leading to * high Lflei» index’
{mean number of fleas per rat). In the hot, dry
weather, fleas do not thrive and the transmiision of
infection Ls interrupted.
The studies of the various governmental Plague
Commissions in Bombay, during the eaily years of
the twentieth century, helped to clarify the
epidemiology of plague. It was found that plague
fiioduced epizootics First in Rattus rton'tgicus
(newer rut). When their numher dwindled, the
disease passed to the domestic rat, R. raffus, It was
from the domestic tit that the infection spread to
human beings.
Two natural cycles o f plague exist, the domestic
and the wtEd. The term \rrban or domestic plague'
refers to plague that is intimately associated with
human beings wild rode nth living with them,
possessing a definite potential for producing
epidem ics. 'Wild or sylvatic plague' occurs in nature
and in wild rodents, independent of human beings.
'I Tic rodents invoivied vary in di fferent regions. Over
.2t)0 Species and Subspecies are involved. In Western
I ISA prairie dogs, ground squirrels, wood rats and
mice arc found infected. In the endemic areas of
the USA, cuSes- of human plague have occurred
following contact with wild animals, and even with
domestic carnivores, particularly pet cats. In java,
[he field lit is the reservoir. In India,, [he gerhil
C o p y rig h te d m aterial
(Tstcru indict) and the bandicoot arc infected.
Human infection may occur during skinning and.
handling of carcasses of infected wild animals.
Carnivores, including c-atH and Jogs can get infected
by eating infected rodents or through their fleas.
Clink-nil plague is seldom seen in dogs, but may develop
in cats. Human infection from inhalation of respiratory1
droplets from infected cab his been reported.
In enzootic foci, plague may persist for long
periods. Infected fleas may survive for over i year,
The bacilli Can remain alive H id even multiply in
the soil of abandoned rodent burrows. They can
infect new rodents that may reoccupy such burrows,
This may account for the long period of quiescence
and Subsequent re-emergence characteristic of
plague. Attenuated strains of plague bacilli have
been isolated from natural foci. T h ey may regain
virulence when plague becomes active. Eradication
of plague is an unlikely prospect as it is a disease of
the earth— of rodents that live in burrows and of
the fleas that live on them . O nly when hu man beings
or domestic animals trespass on these natural foci
do human infectious set in.
In the 199Qs, there has been a re-emergence of
plague in countries where it had ceased To be
noticed for many years. This has happened in the
developing and the developed countries-—India and
China in, Asia, Malawi and Zimbabwe in Africa,
the erstwhile USSR in Europe and in the USA.
Plague bacillus strains carrying plasmid borne
resistance to multiple antibiotics were rejiorted from
Madagascar in 1995, These have the potential to
spread and pose a great threat.
The laboratory should
Ikj able to diagnose plague not only in humans, but
in rodents also, as timelv detection o f infect ion in
rats may help to prevent epidemic spread.
A rat which died of plague may cany infected
fleas and should he handled with care. Pouring
kerosene oil over the carcass is a simple method of
eliminating the fleas. In the laboratory, the carcass
should be dipped in 3% lysol to destroy
ectoparasites.
During epizootics, it is easy to diagnose plague
in rst£. Buboes ale usually present in the cervical
region. They are hard and can be moved under the
skin. t>n section, the bubo may show congestion,
hemorrhagic points or grey necrosis. Smears from
the bubo stained with methylene blue show the
bipolar stained bacilli. The fluorescent antibody
technique may he of use in identifying plague bacilli
in the Impression, films of the tissues. Bacilli in
bubo show considerable pLeontorphism. The liver
is mottled, with red, yellow or grey stippling. The
spleen is enlarged, and moulded over the stomach,
with grannies or nodules on the surface. A
c haracEeristic feature is pleural effusion which may
he clear, abundant and straw coloured nr, less often.,
bloodstained, Bacilli may be demonstrated
microscopically in spleen smear? and heart blood-
Culturcs may be made from the buboes, spleen,
heart blood and particularly, from bone marrow in
decomposed carcasses.,
In badly putrified carcasses, microscopy and
culture may not be successful. The putrificd tissue
rubbed on the shaven abdomen of a guinea pig can
infect the animal. Diagnosis may also be established
by demonstrating the F-I antigen by itnmno-
flumcscent staining.
In human bubonic plague, the bacilli may he
readily demonstrated in buboes by microscopy,
culture or animal inoculation, Blood cultures, are
often positive.
In pneumonic plague, the bacilli can be
demonstrated in the sputum by microscopy, culture
or animal inoculation.
Serological tests are sometimes useful in
diagnosis Antibodies to the F-I antigen may be
detected by passive hemagglutination. Rise in Litre
of antibodies in p i red sera or titre of 12S or above
in a single scram sample can be considered positive.
IgG and IgM ELISA tes-t-^ have been developed.
PC R iai rapid and sensitive method for presumptive
diagnosis of plague in clinical material and fleas.
In the prevention of domestic
plague, general measures such as control of Ilea-.
C o p y rig h te d m aterial
Hidden page
Motility at 22 CC
Growth on M uConb/s agar
AfId from sucrose
Acid from maltose
Indole
Oxidase
Urease
Onotlhirie decarboxylase
Y, CJTfefot^ffrL'j has been isolated from a wide
range of domestic and wild animals and, in recent
years, ls increasingly being reported from human
clinical material. It produces three types of disease
in human beings. The fnSI type occurs in young
children as selM im ifcd gastroenteritis nr
enterocolitis which may be either inflammatory or
noninflammatory.The second is mesenteric adenitis
and inflammatory terminal ileitis in older children
that may mimic appendicitis. The third category Lh
a systemic disease typically in adults,, often
characterised by bacteremia, meningitis, arthralgia
or erythema nodosum. Persons belonging to HI .A
- E 27 group are prone to develop reactive arthritis.
A group of related bacteria isolated from
hemorrhagic septicemia in a variety of animals and
birds had, in the past, been named according to
their species of origin—T . Iwvfsi^JCrCa, jfcpjstpNed,
Jidsepfica, e1r. Though thev show some degree of
host s p e c ific itr , they arc so alike in either respects
that they axe now considered strains of a single
species designated P. inuhocidk
P m u/mciJa is a non [noli le. G rain negative
bacillus generally resembling Tfttdud but differing
in being oxidase positive, producing indole and
failing to grow on M acConkcy’s agar.
The bacillus is often carried in the upper
respiratory tract of a variety of animals such as dogs,
cats, rats, cattle and sheep. It may sometimes occur
as a commensal in the human respiratory tract also.
Human infection is rare hut may occur following
animal bites or trauma. The clinical manifestations
may be local suppuration following animal bites
{wound infection, cellulitis, ahscess, osteomyelitis),
meningitis following bead injury, respiratory tract
infection [pneumonia, bronchitis, sinusitis) or
appendicitis and uppendidal abscess.
The bacillus is sensitive to tetracycEine and
streptomycin and most strains to penicillin as well.
^fhis is tbe causative agent of tularemia,, a disease
of rabhits and other rodents. Originally described
mTulare county, California. Infection is transmitted
by tieki and several other arthropod vectors. Hum,in
infection may occur by direct contact with infected
rodents such ss rahhifs or through ticl; bites. It can
also be acquired hv ingestion of contaminated meat
OT water and inhalation of infective aerosol".
It is a minute, capitulated, nonmotille Gram
negative bacillus, about 0.T-0.7 pm * 0.2 gin Lit
SI7jC. It resemhles mvcoplasma in being filterable
and in multiplying by filament formation and
budding, besides binary fission. In infected animals,
it acts as an intracellular parasite, being found in
feign: masses inside the liver and spleen cells. It has
fastidious growth requirements and special media
C o p y rig h te d m aterial
333 * Textbook (rf Microbiology ►

such as Francis blood dcmosc cystine agar have ro
luLemployed for its LHfJacior. Minute rra.rtSp,Lient
colonics appear after incubation lor 3—5 days.
Srrairsof S.fu/srnisj's have been subdivided info
biotypes based on their virulence and
epidemiological beliaviour. Highly virulent strains
arc found only in N. A merica, while strains of law
virulence arc seen in Europe and Asia also-
In human beings, tularemia may pre-cut as a
local ulceration with [ymphadcniirb, a typhoid like
fever with glandular enlargement or art influenza
like respiratory infection. The ■.Incase may also he
waterborne, as a result of water pollution by the
\lTli .I of infected rodents. The bacillus is highly
infectious and laboratory infection has been quite
common. Diagnosis may be made by culture or by
inoculation into guinea pigs or mice. Agglutinating
,LOI.bodies may be demonstrated in sera from
patients.
An attenuated vaccine is available which can
be administered by scarification to persons who arc
subject to high risk of infection,

Fu rth er R m Jm f
Brubaker 11K. 1991. Factor ■■| i- im_■infccritMis caused by yersiniae. l i M icrobiol Rev. 4:309.
j .-i

Butler T. 1994. Yersinia .nfet nons. CJ.n /nihct£J.i 19:655.

Campbell andJM Hughes 1995. Plague in India. Ann Inr Med. 122:155.
Cover TL and RC Aber 1939. Yersinia entenoculirica. .Vew£ngJ J Med 321:16.
Cage Kl. 1993. Plague. In Topley and Witson'i M icrobiology and M icrobial Infecriom, 9lhedn. London: Arnold.
Ibllitzer R. 1954. Plague. WHO Monograph series Ho. 22.

C o p y rig h te d m aterial
 Haemophilus
Thegenus Haemophilus wntiiit! amall* nonmoulc,
nonsporing, oxidase positive, pleomorphic, Gram
negative l^j.ni]]i that arL' parasitic nn human beings,
or animals. They arc characterised by their
requirement of One or both of two accessory growth
factors (X and V) present in blood (Haemophilus,
meaning blood loving).
Pfeiffer (1892) observed that a small. Gram
negative bacillus was 'constantly present' in the
sputum of patients from the influenza pandemic of
1 8 8 9 -9 2 and mistakenly proposed this as the
causative agent of human influenza. Thin a m t to
he known as the 'influenza hacillus’ (Pfeiffer^
bacillus), later renamed Haemophilus influenzae,
The causal relationship between this bacillus and
human influenza could not he subtitanriatal and w
finally disproved when Smith, Andrcwes and
Laidlaw (1933) isolated the influenza virus.
//, influenzae is a small (1.0 urn
n 0.3 pm), Gram negative, nonmotileTnonsporing
bacillus, exhibiting considerable plcnmprphlsrn
(Fig. 36.1). In sputum, it usually occurs as clusters
of coecobacillarv forms, while in the CSF from
meningitis cases, long, bacillary and filamentous
forms predominate. Cells from young cultures
(1 8 -2 4 hours) arc usually coccobacillary, while
older cultures are distinctly pleomorphic. Strains
isolated from acute infections are often capsulatcd.
The bacilli are relatively difficult to Stain.
Staining for 5-15 minutes with I .oeffltr's methylene
blue or dilute carbol fochsir gives good results-
Thc bacillus has
fastidious growth requirements. The .accessory
growth fectors, named X and V, present in blood
are essential for growth. The heat stable X factor
is hem in or other porphyrins require*! for the
synthesis of cytochrome and other heme enzymes
such ; ls catalase ,ir.il peroxidase involved in acrnhic
respiration, The X factor is not required for
anaerobic growth, The V factor was so named
because it wss originally thought to be a bacterial
vitamin. It is heat labile being destroyed at 120 °C
in a few minutes. It is present in red blood cells
and in many other animal and plant cells. It is
synthesised by some fungi and bacteria (for example.
Staph, aureus) in excess of their requirements and
released into the surrounding medium. The \r factor
is a coemryme, nicotinamide adenine dinudeolide
(N A D ) nr N A D phosphate (N A U P ) which acts
as a hydrogen acceptor in the metabolism of the
cell.
It is aerobic but grows anaerobically also. The
optimum temperature is J7 D C . It docs not grow
below 20' C. borne strains require 10% COr It
grows on hlotxJ agar hut growth is scanty, as the V
factor is not freely available, being imprisoned inside
the red blood cells. Growth is, therefore, better if a
source of the V factor is also provided. When Staph,
aureus is streaked across a pUtc of blood agar on
which a specimen containing //, influenzae has
been inoculated, after overnight incubation, the
colonics of / / . influenzae will be large and well
developed alongside the streak of staphylococcus,
and smaller farther away. Tins phenomenon is called
satellitism, and demonstrates the dependence of
C o p y rig h te d m aterial
 l i M i K w o n t3ie V factor, which i$ available in
high concentrations near tht staphjdoccxxsd growth
and only in smaller quantjtieti away from it. Tiiia k
i routine test in clinical bacteriology for the
idcntificutioji o± //. influenzae (Fig 16.2). It is,
however* not very specific as ir will also be positive
with other V factors requiring hemophili as well
as with occasional strains of ncisscriae and
diphtheroids.
When blond agar is heated to 8 0 -9 0 or
boiled for a few minures (bolted blood agar), the V
factor is released from within the eiyihrotyteH and
hence these media are tiU|?erior lo plain blood agar
for grown ng 11. inStutnzae. Clear tra nspa rent med i-.i
may be prepared by boiling and filtering a mixture
ot blood and nutrient broth (Lcvinrhal's medium}
Oi by adding -a peptic digest of hlood to nurricnr
□gar (Fildes agar). Fildes agar is best for primary
iibhtion of H. iafiuetm t and gives a copious
growth, Capsularcd strains produce translusccrt
trsEonie^ with a distinctive iridescence on Levin thal's
agar,
. Glucose and Xylose
are fermented! with acid production but not lactone,
sucrose and mannitol, Catalase and oxidase
reactions are positive. Nitrates are reduced to
nitriles, Eight hiotypes have been identified on the
buHis of indole production, unease and ornithine
decarboxylase activity. Blorypc i is most Frequent! V
responsible for meningitis.
H. influenzae is a delicate
bacterium, destroyed by heating (55aC for 30
minutes}, refrigeration (0-*l T ) h drying and
disinfectants. In culture, the cells die within two or
three days due to autolysis. Cultures may be
preserved for about a month on chocolate agar
slopes in screw capped bottles, For longterm
preservation, the culture mas-he Ivophiliscd-
There are three major
surface antigens - the capsular polysaccharide, the
l t , '
outer membrane proteins (OM P) and
lipooligosaccharide (LOS).

C o p y rig h te d material
 ■ Haumcphlui »
The minor antigenic determinant of capsulatcd
strains is the capsular polysaccharide based on
which H. jjUj'Jucri^e strains have been classified
by Pittman info six cupsulur types - types a to f.
Typing was originally done by agglutination but
other methods such as Quellung reaction,
lire-, ipilati-uc], coflggluli:iaLio-nhCl E and ELISA may
aIso he u*cd. L' .1 p- nlar typing in o1 m edical
importance as about 95 per cent of H. influenzae
isolates from acute invasive infection* such as
meningitis belong to type b. Diagnostic kits lor the
identification of H. i.'irtyenzae type l"1 (H it) arc
com mere ially available.
The type b capsular polysacchai ide has a unique
chemical structure, containing the pentose sugars
ribos-e and ribitot instead of the hcxnscs and
hexosamines as in the other five Rerntvpes- The
capsular polyribosyl ribitol phosphate (PKP) ariigcn
of Hib induces IgG, IgM and IgA antibodies which
are bactericidal, opsonic and protective. Hib PRP
is therefore employed for immunisation. tlib
capsular anligen shows cross reaction with the
capsular anrigens of some Gram positive and ( iram
negative bacte du­
ff. influenzae strains lacking a capsule can not
be typed and are called 'nontypahle strains. Next
ro J lib, the nontypablc strains arc the most relevant
in clinical infections.
The outer membrane protein antigens show
considerable variation. OMP antigens of Hib
have been classified into at least 13 subtypes.
H. influenzae lipooligosaccharides are antigenicaily
complex. OMP and LOS subtyping may be of
epidemiological value.
H. influenzae in the firet free lining organism
whose eomplen- genome has been sequenoed.
Pithcqfen icity; H. influenzae is an exclusively
human pathogen. It .s not naturally pathogenic for
animals but imraperitoneat inoculation of large
doses is fatal in mice, guinea pigs and rabbi;s.
Diseases due to H. influenzae may be considered
under two groups, invasive and nor imvasi vc diseases.
In the fust group, the bacillus acts as a primary
335
pathogen, causing acute mvasi'.x infections. The
bacilli spread through blood, being protected from
phagocytes by their capsule. Haemophilus
meningitis is the most important infection in this
group, others being laryngocpiglofitis,
conjunctivitis, bacteremia, pneumonia, arthritis,
endoCardiLis and pericarditis. These infections are
usually seen in children and are caused by the
capsulaied strains, type b accounting for must cashes.
In the second group, the bacillus spreads by local
invasion along mucosal surfaces and causes
secondary or superadded infections, usually of the
respiratory tract. These include otitis media, sinusitis
and exacerbations of chronic bronchitis and
bronchiectasis. These are usually seen in adults and
are often caused by rhe noncapsulated strains.
M ening.ilis: This is the most serious disease
produced by H. influenzae with case fatality rates
up to 90 per cent in the untreated. The bacilli reach
the meninges from the nasopharynx through the
bloodstream. The disease is more common in
children between two months and three years of
age. Tbi-; age ini' ide nee has been correlated with
the absence of bactericidal anti-PRP antibodies.
Older children develop immunity as a result of
subcliciical infection. It has been reported that in
the trop:cs, non-type b strains may be responsible
for meningitis more often than in the temperate
zones.
L ary n g o ep u g L o ttith ( c r o u p ) : This i> an
acute inflammation of the e[i:. c^lotT iw ith
obstructive laryngitis, seen in children above two
years. Untreated eases may be fatal winlvjn hours,
Tracheostomy is often necessary to relieve
respiratory obstruction caused by the grossly
enlarged uvula. This com!irion is always associated
with bacteremia and blood cultures are usuallv
positive.
Pneum onia1: Haemophilus pneumonia typically
occurs in infants and is accompanied by empyema
and sometimes meningitis Jv well. In older
children and adults, the picture is of Lobar
pneumofiiL W1 ■.lie these are priman jnfections due
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Hidden page
 * H e m o p h ilu s *
the upper respiratory tract. Infection i-. transmitted
by the respiratory route. Carriage in the upper
respiratory tract is common pamculirii in young
children but such strains arc usually noncapsulated
and not responsible for acute invade infection.
Immunity is type specific. As the large majority
of Rerinus infections are caused by type b >Li .1 i: i
active i 1pin nr. Nation with Hib REtP vaccine is
indicated. Purified PRP ii immunogenic in older
children and adults. However, in Common with
other polysaccharide anngens, FRP is poorly
immunogenic in children below two years. Its
immunogpriT- icy can be improved by coupling with
protein carriers like diphtheria and tetanus toxoids
or meningococcus outer membrane protein. Such
conjugate Hib PRP are available far use in young
children.
Young household contacts of patients with
systemic H. influence infect 1on have Increased risk
of getting hitccted. Rifampicln given lor four diyS
prevents secondary infection in contacts and also
eradicates carrier state.
I I a i HOF n i f L s A 1-.m P m s
(itWi-VVrtib bidllui, formerly It itgyptiais)
Even before Pfeiffer described the 'influenza
hacillus1, Koch ( l££3) had observed a small bacillus
in conjunctivitis cases in Egypt. It was first cultivated
by Weeks (1857) in New York and came to be
known as Kuch-Weeks bacillus. Recent DNA
Studies have shown that the bacillus iS identical
with non-capsulared H. nithicni-^c. Therefore,, the
farmer H. acgrpticiix bos lost its separate species
-status and Ls now considered as a biotype ofri.
1.'m 1- If is worldwide in distribution and causes
a highly contagious form nf conjunci iv iris ('pink
eye'). Ic I-; especially common in the tropic? and
subtropics and may occur in epidemic forms. It
responds to lru:a! sulphonami'les or gentamicin.
It has also been identiried as the causative agent
of Brazilian purpuric fever (B P F h in which
conjunctivitis proceeds to a fulminant septicemia
in in fin rs and children with high fatal iiy. First
3 37
retognsed in Brazil in J9SJ4, E P F is now endemic
in South Ameiuia.
H a e m o p h il u s D u <i r e y i
Ducrey (1890) demonstrated this bacillus in
chancroid lesions and by inoculation into the skill
on the forearm, was able to transmit the lesion
through several generations.
Chancroid or soft sore is a venereal disease
characterised by tendernonmdurated irregular uleefS
on the genitalia-'Litis infeed on remains local iscd*
spreading only to the regional lymph nodes which
arc enlarged and painful Autoinoculation lesions
may be produced by contact. There is no immunity
following infection but 1 hypersensitivity results,
which can be demo ns trated by intradermal
inoculation of killed W l:111i.
lir ducreyi is a short, ovoid bacillus (1—1.5 pm
* 0.6 mm) wnh a tendency to ocean in end to end
pn1rs or short cha ins. If ir Gram nega I ive but often
may appear Gram passive and frequently shows
bipolar staining. The bacilli may be arranged in
small groups or whorls or in parade] chains giving
a school of fish ur 'rail road track' appearance.
Primary isolation is difficult. It can be grown
on fresh clotted rabbit blood- Smears made after
24-48 hours incubation show tangled chains of
bad Hi. Tt may also W grown on the chonoallanuuc
membrane of the chick embryo. On chocolate agar,
enriched with isovitaiex and fetal calf serum, and
containing vancomycin as a selective agent, H.
ducreyi Forms small, grey translucent colonics alter
incubation at 35 DC under 10 per cent COj and
h:gh humid1ly in 2—8 days.
The spc'ics is antigenerally homogeneous and
cultures mav be identified by agglutination with
the antiserum. Intradermal!noculatioo of the culture
into rabbits produces a local ulcerative Ic-ion.
H, ducreyi is susceptible to sulphonnmidcs and
many antibiotics- Cases resistant to Rulphonamides
and tetracyclines have been reported. Erythro'
myein. corrimoxa-zole, dproflovacin or ceftriaxone
may be used for treatment.
C o p y rig h te d m aterial
Hidden page
 Bordetella
The genus finrdetelta is- named after Jules
Bordet, who n]nng with Gcngou identified the
$Linill] nvoul bacillus causing whooping cuugh, li]
the sputum of children suffering from the disease
(1900) and succeeded in cultivating it in a
complex medium (1906). The bacillus is now
known as RfmJetelLi pertussis (jxtjrussfs meaning
intense cough). A related bacillus, Burt),
paraperrtiiiii was isolated from mild cases ot
whooping cough (1937). Bord, bronchiseptics
originally isolated frum dog’s with broncho-
pneumonia (1911) may occasionally lilfeer human
beings, producing a condition resembling pertussis-
Ir ha? been suggested that BvfiL brooch fsepfrea
represents the ancestral form from which the other
two species have evolved- "ITe bninth member uJ
the gemLH is BonJ. JtHIiffl which causes respirators
disease in turkeys.
Uorrf- pertussin is a small, ovoid
cuccubaeillus (mean length 0 .i pm). In primary
cultures, cells a r c o f u n ifo rm size and sh ap e but OIL
subculture they may become lo n g e r an d thread like.
It is nnrLinotile and nonsporing, It is tap tallafo d but
te n d s to lose the capsule on repeated cu ltiv a tio n .
The cap su le can lie d e m o n s tra te d by special stains
bu t docs not swell in the prese nee o f the antiserum.
In cu ltu re film s, th e bacilli te n d to be arranged in
loose dumps, with clear sp a ce s in b etw een giving
a ‘thumb print' appearance. Freshly isolated strains
of iin r d . p e rtu s s is have fimbriae.
It is Gram negative. Bipolar metachromatic
granules may be demonstrated on staining with
tnliiidine blue.
11 is aerobic No
growth occurs anaerobic ally. It grows best ar 3 5 -
36 *C
Complex media are necessary for primary
isolation. The medium in common use is the
Bordet-^Gcngou gjiyocrinc-potatu-blnod agar, 13Umd
is required not to pm idc additional nutritive factors
bur rather to neutralise inhibitory materials formed
during bacterial growth. Charcoal or ion exchange
resins incorporated in culture media may serve die
same purpose. Charcoal blood agar is a useful
medium. Ic does not grow on simple media like
nutrient agar.
Growth is slow. After incubation for -IS—"72
hours, colonies CHI Bordet-Gengou medium ate
small, dome shaped, tmoorh, opaque, viscid, greyish
white, refractik and glistening, resembling 'bisected
pearls’ or 'mercury drops Colonies are surrounded
by a hazy zone of hemolysis. Contlueni growth
present a n ‘aluminium paint' appearance.
It is biochemically
inactive. It docs nor ferment sugar*, form indole,
reduce nitrates, utilise citrate or split urea. It
produces oxidase and usually catalase also.
It is a delicate organism, hting
killed readily by heat (55 for 30 minutes), drying
and disinfectants. Rut unlike H. mtftrenzK it retains
viability at low temperatures (0-4 PC).
Outside the body, Bord. pertussis in dried
droplets is said to survive for five days on glass,
three days on cloth and a few hours on paper.
C o p y rig h te d m aterial

Several antigenic fractions ind putative
vimlcncc factors have teen described bur their rck
in the pathogenesis of pertussis remains to be
clarified- They include the following:
J. lSofdetflllae pluses? genus
specific and species specific surface agglutinogens
associated with rhe capsular K antigens or
fimbriae. By agglutinin absorption tests, 14
agglutinating factor* hav-e been iden t iMed. Factor
7 is common to all three mammalian species of
bull'drtelke. Factor \2 is specific for Word.
brQTichis-zptU.1 and Factor 14 for Bond,
parapertussis. Factors 1 to 6 arc found only in
strains of Bond. pCfTU5rishall of which earn- Factor
1 and one or more of the other factors. Bordetdbe
are djunfled into various types based on the
agglutinogens they carry. A h strains causing
infections belong to types I, 2 and,!, it is essential
chat pertussis vaccine strains should have factors
I, 2 and 3 . Factor specific antibodies arc present
in the sera of convalescent and immunised
persons. Agglutinogens promote virulence by
helping bacteria to attach to respiratory epithelial
cells. They arc useful in seiotyping strains and in
epidemiological studies.
J. This is present only in
Word. pertussis. Ic plays in important role in the
pathogenesis ot whooping cough. P I’ is expressed
on the surface of the bacillus and secreted into
the surrounding medium. The toxin exhibits
diverse biological and biochemical activities,
which formerly had been believed rn be caused
by diiferent: substances that had been named
accordingly. Examples are the tymphocytOsit
producing fa tter or L P F causing profound
lymphocyto&is in pertussis patients as well as in
experimental animals; and two effects seer only
Lit experimental annuals, but not in patients, such
as the histamine sen si rising factor or H SF
responsible for heightened sensitivity to histamine
iti experimental animals, and rhe jsfcf activating
prtftcin or 1AP inducing excessive insulin secretion
by the pancreatic islet cells. It is now known that
all these arc manifestations of the pertussis toxin,
PT is ;L 117,000 molecular weight hexamtr
protein composed of six subunits with a]i A-U
structure (the A portion being the enzymatically
active moiety and fl the binding component). It
can be toxoided, P I’ toxoid ia the major component
or' acellular pertussis vaccines. Antibody to PT
Can protect mice against luinmasal, IntruperiturtcaJ
or intracerebral challenge.
3. This is one
(it the three hemagglutinins produced by Word,
pemi.iSj'.s, the others being PT and a lipid factor.
Purified FH A appears as a filamentous structure
in the- electron |tlicWffi|K and heme the name.
lr is present on the bacillary surface and is readily
shed, ll adheres to the cilia ot respiratory
epithelium and to erythrocytes. Resides facilitating
adhesion of Bond p trtu ssii to respiratory
epithelium, FHA and PT hemagglutinins also
promote secondary infection bv coating other
bacteria such j -h HjeiliopJijJuS itifluentae amt
pneumococci and assisting their binding to
respiratory epithelium. This phenomenon has been
termed piracy o f adhesins.
Antibody Co FHA OKI protect mice against aerosol
challenge. FHA is used in acellular pertussis
vaccines along with PT toxoid.
4. All mammalian
bordctdlac but not Bard- tviam product adenylate
cyclase. At least two types of AC arc known, only
one of which has Ilie ability to enter target cells
and act as a toxin. This is known as AC toxin
(ACT). It acts by catalysing the production of
CAMP by various types of cells.
5. It is a cytoplasmic
protein present in all bordctcUac. Ir is inactivated
in 30 minutes ar 56 ^C. It is dermonecrotk and
lethal in mice. Its pathogenic role is not known,
b. Twchad cytotoxin ( T C T ): It is a low molecular
weight pepiidoglycan po educed by all bordetellan.
It induces ciliary damage ill hamster tracheal ring
cultures and inhibition of 1>NA synthesis in
C o p y rig h te d m ateri
 epithelial ccli cultures. fts rule in disease i* not
known.
7. or the heat itihle
toxin is present in atl hordetetlae and evtiihifR
features of Gram negative bacterial endotoxins, ]t
is present in the whole eell perrussis vaccine but
is not considered to be a protective antigen.
8. Pertactin is an outer membrane
protein (OMP) antigen present in nil virulent
strains of B . pertussin. Mice immunised with
pcrtactin resist respiratory challenge with rhe
bacillus. Antiltody L-:;- pertaetin can be seer in the
blood of children after infection or immunization.
Pen an in is included in acellular pertussis vaccines.
lio r d pertussis undergoes a smooth
to rough variation. AH fresh isolates are in the
smooth form (Phase I). On suhculture, they
undergo progressive loss of surface antigens, anti
pass through phases II and III-, finally becoming
phase IV which is the rough, avirulcnt form,
A reversible change in the capsular antigen has
been described as modulation.'- The bacillus may
occur in one of three potential "modes', X, 1 and C,
each of which has a characteristic surface antigen.
X, I and C refer to the colour of the confluent
colonies on the Bordet“ Gcngou medium - X fbr
AantAic (yellow), C for cyanic (blue) and 1 for
intermediate. Modulation is influenced by the
nature of the culture medium. On the Bordet-
Gengou medium, fresh isolates always occur in the
X mode.
B o rd , p ertu ssis is an obligate
human parasite but infection can he produced
experimentally in several species of animals, the
white mouse being most often employed, lntranasul
inoculation in mice induces a characteristic patchy
interstitial pneumonia, histologically resembling the
human disease. Intraperitoneal inoculation of large
doses is fatal due ta toxemia. Intracerebral
inoculation causes a fatal infection. Immunised mi cl
arc protected. This forms the bads for the
intracerebral mouse potency assay for pertussis
vaccines.
In human beings, alter an incubation period of
about 1-2 weeks, the disease takes a protracted
course comprising three stages - the catarrhal,
paroxysmal anti convalescent - each lasting
approximately two weeks, "Hie onset is insidious,
with low grade fever, catarrhal symptoms and a dry
ini taring cough. Clinical diagnosis- in the catarrhal
stage is difficult. This is unfortunate as this is the
stage at which the disease can be arrested by
antibiotic treatment. This is also the stage of
maximum infect ivity. As the catarrhal stage
jdviLTicEH to the paroxysmal stage, the cough
increases in intensity and comes on in distinctive
bouts. During the paroxysm, the patient is subjected
to violent spasms o f continuous coughing, followed
hv a Long inrush of air into the almost empty lungs,
with a characteristic whoop (hence the name). The
paroxysmal stage is followed by convalescence,
during w hich the frequency and severity o f
coughing gradually decrease.
The disease usually lasts 6-8 weeks though in
some it may be very protracted. Complications may
be 1) due to pressure effects during the violent bouts
o f coughing (subconjunctival hem orrhage,
subcutaneous em physem a), 2) respiratory
(bro ncho -pn eum o nia, lung collapse), or 3}
neurological (convulsions, coma). Respiratory
coni p Licat ions are self lim ited, the atelectasis*
resolving spontaneously hut the neurological
complications may result in permanent sequelae
such as epilepsy, paralysis, retardation, blindness or
deafness.
The infection is limited to the respiratory tracr
and the bacilli do not invade the bloodstream. In
the initial stages, the bacilli arc confined to the
nasopharynx, trachea and bronchi. Clumps o f bacilli
may he seen enmeshed in the cilia of the respiratory
epithelium. As the disease progresses, inflammation
extends into the lungs, producing a diffuse
bronchopneumonia w ith desquamation of the
alveolar epithelium.
Blood changes in the disease are distinctive and
helpful in diagnosis. A marked teucacvCosis occurs,
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Hidden page
Immunofluorescence is useful in identifying the
bacillus in direct smears o f clinical specimens and
o f cultures. T h e d iffe re n tia tin g features of
bordetelke are listed in Table 37.1.
Serological diagnosis is not helpful. Rise in
antibody dire may be demonstrated in paired serum
samples by agglutinarion, gel precipitation or
complement fixation testa. As antibodies appear late,
the second sample o f serum should be collected
some weeks after the onset o f rhe disease.
Demonstration of specific seCretOrY Ig A antibody
in nasopharyngeal secretions by E L IS A has been
proponed us ;ldiagnose method in culture negative
cases.
Preventing the spread o f infection
by isolation o f cases is seldom practicable, as
inactivity is highest in the earliest Stage o f the
disease when clinical diagnosis is nor easy.
Specific immunisation with hilled Bord. perfurars
vaccine has been found very effective, It is of utmost
importance to use a smooth phase 1 strain for
vaccine production. The method of inactivation
should be such chat antigenic potency Is unaffected.
Detoxication with 0,2^i merthiolaco during several
months' storage at 4 "C Kan been recommended as
a satisfactory procedure.The alum absorbed vaccine
Motility - -
Growth on nutrient a^ar
G ivAvrh on Bordet-Gengou 3-6
medium {days)
Urease
Nitrate to tii trite - -
Citrate utilisation - V
Oxidase * -
Toxins:
HLT and TCT
ACT
PT
V * Variable
produces better and more sustained protection and
lew reaction than the plain vaccines. Pertussis vaccine
is usually administered in com bination w ith
diphtheria and tetanus luxoid {triple vaccine). Not
only is this more convenient, but ZJorJ. pertussis
also acts as at] adjuvant for the toxo]ds, producing
better antibody response.
In view of the high incidence and severin' ol
rhe disease in the newborn, it is advisable to start
immunisation as early as possible. Three injections
at intervals o f 4 -6 weeks arc to he given before the
age o f sot months, followed by a booster at the end
o f the first year o f life.
Children under four years who are contacts of
patients should receive a booster even if they had
been previous]!' immunised. They should also
receive chem oprophvlaiis w ith erythrom ycin.
Non inn tp united contacts should receive
erythromycin prophylaxis for ten days after contact
with the patient has ceased. Pertussis vaccination
ttiuv induce reactions ranging from local soreness
and fever, to shock, convulsions and encephalopathy.
Provocation poliomyelitis is a rare complication.
Factors contributing to toxicity or pOEtTOcdluJ
encephalopathy have not heen defined. The latter
complication is estimated to occur in one in 5-10
f
* +
1-2 1 1
■fr ■P -
# -
* V
# *■
* ■* +
* * -
- - -
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m illion injectlonR. E stim a te d neuriilflgicil
complications o f natural disease have ranged front
1 5 - 1 4 per cent in hospitalised cases; i\ third of'
these recover, a third have sequelae and a third die
or have severe delects.
[f severe complications-SLLL-h as encephalopathy,
■seizures, shook or hyperpyrexia develop following
the vaccine, subsequent doses of the vaccine are
cciTitrsindiiiatLMl. Routine pertussis vaccination is not
advisable after the age uf seven years as adverse
[“dictions are likely ami the risk of severe disease is
low. Acellular vaccines containing rhe protective
components of the pertussis bacillus (PT, Ell A,
agglutinogens 1r 2, .1), fin-E deve loped in Japan, are
now used in some other countries also :lhrhev cause
far fewer reactions, particularly in older children.
Both whole cell and acellular vaccines have a
protection rate of about % j^er cent. With wltolc
cell nCQlU, the protection declines hi SOper cert in
about five yenrc and is absent after 12 ycare, I’hc
duration of protection with acellular vaccines is riot
known. Even fully immunised subjects may develop
pertussis but the disease will be very mild in them.
B o n l . pertussis is susceptible to
several a n t ib i o tic s {except p e n ic illin ) blit
antimicrobial therapy is beneficial oniy if initiated
w ith in the first ten days o f llie disease.
Ervthromveiu or one of the newer macralides is
the drug o f c h o ic e . C h lo r a m p h e n ic o l and
cotriTTicoatzolc arc also useful.
This is an infrequent cause of whooping cough.
The disease is mild. The jKTtussls vaccine does not
protect against Bond- panpertussis infection.
T liiy is m o tile hy p c ritrlc h a te fla g e lla . It is
lUitigenically related to fiord, pertussis and Brucetti
abortus. It occur naturally in the respiratory tract
of several species o f animals. It has been found to
cause a very small proportion (0.1 per « n t ) o ( cases
ijf whooping cough.

Cherry JD cf al. l9Sii. Report of chc usk hirec ewi Pcriusiiv Ffciiiii 8t:fl39.
Editorial. 3992. PL-rruscis. adults, infants and Iil-ilIc Lancer, 3.19:526,
Friedmin til •• 39$?. The i Ukl-jm: .i r.aI diagnostic ni((bi)dfc <'I'Jrt Miobbirf fiev 1:365.
Gustaffson I. et al. 19%. A controlled tnal of acellular pertussis vaccines. A’ettr tn gi /Afed. 333:349.
Pittman M. iyH4.Tlie concept of pertussis as a rosin mediated duea.se. Pediair Infcc r Dis. 3:4t>7.
Weiss AA and EL f levrietr l98fc. Virulence factors of Bordetelh pufnoit Arm RcvM itrobial4U.661.
1Hewlett EL V997. Pertussis: Current concept? of pathogenesi?. H c t ia t r ic fn fe c T D js. J 16:579

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 Brucella
The genus B ru c e lla consists uf very small,
ncmmotile, aerobic, Gram negative Coccobacllili that
puw poorly on ordinary media and have little or
no fermentative powers, They arc strict parasites of
animals and may also infect humanj..
Brucellosis is a zoonosis, primarily affecting
goats, she up, cattle, buitaloes, pigs anJ other
an imals and tran smitted to hu ma ns hy con tact wi th
infected animals or through their products. The
human disease was recognised along the
Mediterranean littoral from very early times and
has been known under various names such as
Mediterranean fever, Malta fever and undul.inf lever
A British army doctor, David Bruce (1886)
isolated a small microorganism from the spleen of
fatal cases in M alta and transmitted the disease to
monkeys experimentally.This was. named BmcnefJ.ii
mcJj'fcnsj's (Brucella after JJjucc, mclitcnaia after
Melitat the Roman name lor Malta), A Maltese
bacteriologist Zarnmit (1905) showed that Br.
nuriircnsjFwas transmitted to humans by psat's milk.
Following this, the disease was eliminated from
British soldiers by prohibiting diem from using
gnat's m ilk and m ilk products, while the disease
remained undi mi rushed in the civilian population,
w hich continued to use them , Bang (1 8 9 7 )
described Br. aburlUt, the cause o f contagious
abortion in cattle. T h e third major species in the
genus, Be aiu'i was isolated by Traum (1,914) from
pigs in the USA . These three species cause human
brucellemis.
Other species causing animal infections include
Be ranis, isolated from, cases of canine abortion,
fir. ovj.* from abortion in sheep and B r ncofmnae
from desert wood rats. B r eanis may occasionally
cause a mild human disease, but the other [wo are
not pathogenic for humans.
Bruecllae are cotcobucilli or short
rods C,5-0.7 pm * 0.6-L5 pm in size, arranged
singly or in short chains. The cells are so small
that they may be mistaken fur cocci, as was dune
by Bruce who called them Micrococcus m ditcnsis.
In older cultures, irregular forms appeal. They are
no iLcntft ile. noncapsulated ai lJ ftdnspOring. They are
Gram negative and nonacid fast, Bipolar staining
is nor uncommon.
Bmcetlae are strict
aerobes and do not grow anaerobically. B r abortus
is capnophilic, many strains requiring 5-10'k) C O .
for growth. The optimum temperature if 37 *C
(range 20-40 DC ) am! p H fr-6-7.4. They may grow
on simple media, though growth is slow and scanty'.
Growth is improved by the addition of scrum or
liver extract. Liver infusion media were widely used
tor the cultivation of brucellas. The media empkryed
currently arc scrum dextrose agar, scrum potato
infusion agar, irypricase soy agar, or iryptosc agar.
The addition of bacitracin, polym yxin and
cycluheximide to the above media mates them
selective.
In liquid media, growth is uniform, and a
powdery or viscous deposit is formed in old cultures.
On solid media, colonics arc small, moist,
translucent and glistening. Mucoid, smooth and
tough types of colonies appear, associated with
changes in antigenic structure and virulence,
Erytli.ritol has a specially stimulating eifect on
the gmwth o f bruecllae.
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 No carbohydrates Sltl"
ordinarily fermented, though they p o sse t uxitkbvE
capacity Brucellae ate catalase positive^ oxidase
positive (rveepr tor Br. ncotomar and BrQliiwhidi
are negative) and urease positive, Nitrates arc
reduced ro nitrites. Citrate is not utilised. Indole is
not produced and MR and VP rests are negative.
Brucelke arc destroyed by heat at
60 'C in 10 minutes and by l*hi phenol in 15
minutes. Th.ev are killed hv paSTHiri^ation. They
may survive in soil anil manure for several weeks.
They remain liable thr HI day^ in refrigerated milk,
one month in ice cream, four months in butter and
for varying jK-riods in cheese depending on its pH.
They may also survive for many weeks in mem.
They are sensitive to direct sunlight and acid, and
rend to die in buttermilk. Hr. meHtcn^is may terrain
alive for six days in urine, six weeks in dust and ten
weeks m water.
The somatic antigens of
brucellav contain two main antigenic determinants,
A ami M. which are present in different amounts in
the three major species- Br. abortus contains about
20 times A us M: B r meUtonsis about 2(1 times M
as A- Hr sihs has an intermediate antigenic pattern.
Ab&orptio n of th c mi not alinger ic compon cnt from
an antiserum will leave most of the major antiluidy
component and such absorbed A and M
monospecific scr.i arc useful for species
identification hy the agglutination test. The species
identification of brucella strains is not, however, so
fitnightforward and strains are often seen that
behave biochemically as abortus and serologically
as melitensis and vice versa. Species and biotype
identification dejsends on a variety of other factors
besides antigenic structure (Table 38.1).
A n tig en ic eras e- reactions exist between
brucellar and Vr choltf& r and perrons receiving
rhe cholera vaccine may develop brucella agglutinins
lasting tor about three tears. Antigenic cross-
re actio ns also exist with F . coii 0:11b; 0:13 7,
Salmonella serotypes group N (0:30 antigen
Kauffm an anti W h ite ), f V m afrophjhi, Y.
tnreroeoJirica and F tulareiiiis. A superficial L
antigen resembling Salmonella V i antigen has been
described.
Several bacterial
ph;igC' that lv?c the Brucella strains have Hccn
isolated. A ll these phages are serologically similar.
The Tblisi (Tb) phage has been designated as the
reference phage and at R T D lyses only lit, abortus.
Br. suis is lysed at 10,000 K T D , while B r / m Bih k js
is iLot Lysed at all.
Bmediae may be classified into
different species, h^sed on C O , requirements, H ,S
production, sensitivity to dyes (basic fuchsia and
tli ion m), agglutination by KHHUHpedlk sen, phage
lysis and oxidative metabolic tests with urn ino acid?
and carbohydrates. The three major species are Br
mciitttuis, Br. abortus, infecting primarily goats or
sheep, cattle ,tnd swine, respectively. Many biotypes
have been recognised in these species.
Br. Suis strains tliat produce H ,S are krtuWu 4s
'American' strains and those rhat do not a* 'Danish'
strains.
A ll three m ajor species o f
bruccllac arc pathogenic to human beings. Br.
meiitensix is the most pathogenic, Br. ahrirty-T anti
Hr. suis o f interm ediate pathogenicity. T h e
incubation period is usually about 10-30 days, but
may si >meti mes be very prolonged- Humsn i rtforrion
may he o f three types:
1. latent infection with only serological hut no
clinical evidence;
2. acute or Subacute brucellosis; and
3. chronic brucellosis.
Acute brucellosis is mostly due to Br. melititnsis.
(It is LLSunllv known Ifi undulanf fever, but this is
misleading as only some cases show the imdulant
partem.) It is associated with prolonged bacteremia
ami irregular lever. The symptomatology is varied,
consisting o f muscular and articular pun is, asthmatic
attacks, nocturnal drenching sweats, exhaustion,
anorexia,constipation, nenmus irritability and chills.
T h e usual cciniplications are articular, osseous,
visceral or neurological.
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 i Brucella ►
Laboratory methods for d:ngno5i-- include culture,
Htrology and hypenienH 11ivi ty Ccstn.
Blood culture is the most definitive method for
the diagnosis o f brucellosis- Blood is inoculated
.iitn 1 hnttlc f>f trypricase sny broth and incubated
it 37 “C under 5-10% C G r As bacteria in blood
ire usually scanty, large volumes o f blood (5 ml)
should be inoculated. Subcultures are made on solid
media every 3-5 days* beginning on the fourth day,
Growth mav often be delayed and cultures should
not he declared negative in less than 6 -8 weeks.
BACTEC cultures may become po- irive in 5 to 6
days.
The Castaneda method o f blood culture has
several advantages anc is recommended. Here, both
liquid and solid media are available in the same
bottle. The blood is inoculated into the broth and
the bottle 'ncuhatcd .n the upright position. For
subculture, it is sufficient if the bottle is tilted so
that the broth flows over the surface o f the agar
slant. It is agiun incubatedin an upright position.
C olonies appear on the slant. This method
minimises materials and manipulation, reducing
chances o f contamination and risk o f infection to
laboratory workers,
Blood cultures are positive only in about 30-50
per cent o f cases, even when repeated samples are
rested, ifr. inefjrensj'i and Sr. sills are isolated more
readily than Br. abortus. Bone minnow cultures j Leld
a higher rare u f isolation and remain positive long
after the blood culture his become negative.
Cultures may also be obtained from lymph nodes,
cerebrospinal fluid, urine and abscesses, i: present,
and, mi occasion, also from sputum, b nrasI milk,
vaginal discharges and seminal fluid.
As cultures are often unsuccessful, serological
methods are im portant in diagnosis. Several
serological tests have been developed, including
agglutination, complement fixation and E L IS A .
T h e standard agglutination rest (5A 1 ' is
performed most often. Th'.* :s a tube agglutination
test in which equal volumes of serial dilutions of
the pa dent's serum and the standardised antigen fa
34y
killed suspension o f a standard strn' u of Br. abortus)
are mired anti incubated at 37 (. for 24 hours or
50 for IS hours, A litre o f 160 or more is
considered significant- Most patiints with acute
hrucellosis develop ritres o f 640 or more by 3—4
weeks o f illness. Titrcs tend to decline after the
acute phase o f the illness.
Several sources o f error have to he guarded
against, Sera often contain 'blocking’ or
'nonaggluti.iLatinj.'' antibodies. The blocking effect
may somcf mes be removed by prior hcaf ng o f the
serum ar 55 for 30 minutes or by using 4 ^ saline
as the di Iuent for the test- The most fe :-3blc meth nd
for obviating the blocking effect and detecting the
'incomplete' antil>odn"- is the anti globulin {Coombs)
test. A h the prozone phenomenon to high titrcs
(upto 1/640) is very frequent in bruccllos^, ii :s
essential that several serum dilutions he tested- A
positive agglutination test may be produced by
cholera, tularem ia nr yersinia in fe ctio n , or
immunisation. Cholera induced agglutinins mav he
differentiated by the agglutinin absorption test and
also as they are removed by treatment with 2-
m ercapto-ethanol. In order that results from
different la b o ra to ry arc comparable, ir l,s the
practice to express agglutinin titres :n International
Units- This is done byr using a standard reference
serum for comparison.
In brucellosis, both Ig M and IgG antibodies
appear in 7 -1 0 days after the onset o f clinical
infection. As rhe disease progresses, Ig M ant bodies
decline, while tire IgG antibodies persist oi Increase
in citrr. In chronic infections, Ig M may often be
absent and only [gG can be demonstrated. T h e
agglutination rest idcnT:fies m ainly the Ig M
antibody, while both Ig M and IgG fix complement.
The Ig G and Ig A antibodies may act as 'blocking’
or 'nonagglutinating' antibodies. It is thus evident
that the agglut i nat'.i m test is usually po-’ i rivL- i n acute
infection but may be only weakly posirive or even
negative in chronic cases. T h e rebuild o f the
agglutinallion tests therefore have Co be evaluated
carefully. W hile a Inch firm o f agglur'.inns, and
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350 e Texlbook pF MicrDbiaior]y ►

especially demonstration o f a rise in titre, can be
taken .lk diagnostic, even a negative aggluti nation
tost may not exclude the possibility of brucellosis.
The complement fixation test is more useful in
chronic cates as it detects Ig G antibody also.
E L IS A sensitive, specific and can detect Ig M
and Ig G antibody separately Iris therefore useful
for differentiation between the acute and chronic
phases o f brucellosis srnd also for screcr.ing large
numbers o f sera.
Hclayed hypersensitivity type skin testa with
brucella antigens (‘brucellins') ate not useful in
diagnosing acute brucellosis. They parallel the
tuberculin test in indicating only prior sensitisation
with the antigens, and may remain positive for yean*
Brucellin testing may lead to a rise in tine of antibodies.
The methods used for the laboratory diagnosis
of human brucellosis may also be employed for the
diagnosis of animal infections. In addition, bmcellae
may be dem onstrated m icroscopically in
pathological specimens by suitable staining or by
immunofluorescence- Several rapid methods have
been employed for the detection of brucctlo^i-. in
herds o f cattle. These include the rapid plate
aggfuDjTdtfoiT test and the /Jose Bengal card test.
For the detection o f infected animals in dairies,
pooled m ilk samples may be tested for bacilli by
Culture and for antibodies by several techniques. In
the milk, ring test a sample o f whole milk is ir.ised
well with a drop o f the stained brucella antigen (a
concentrated suspension of killed B r abortus stained
with hematoxylin) and incubated in a water bath
a t7 0 <1C for 40-50 minutes. lfantibodi!-s are present
in the milk, the bacilli are agglutinated and rise
w ith the cream to form a blue ring at the top,
leaving the milk unstained. I f antibodies are absent,
no coloured ring is formed and the milk remains
uniformly blue. T h e whey agglutination test is
another useful method for detecting the ;mtibodies
in milk.
P r o p h y la x is ! As the m ajority o f hum an
infections are acquired by consum ption o f
contaminated m !Ik, prevention consists o f checking
brucellosis in dairy animals. In many advanced
countries, thb is achieved by the detection o f
infected animals, their elimination by slaughter and
the development o f certified hnicella-free herd*.
Pasteurisation o f milk is an additional safeguard.
Vaccines have been developed for use in animals.
Br. abortus strain 19 vaccine i=. protective irt cattle.
No suitable vaccine is available for human use.
T runinicn i: The usual regimen is a combination
o f doKycydine for 45 days with streptomycin I M
daily for the first 1 weeks in adults, and in
children cotrimoxazole along w ith rifampiem or
gentamycin.

F u rth e r Hunt!
Corbel MJ. 1W7. Brucellosis, an overview. Emej^.rig Infect Dis. 3:No. 2.
CutW 19^7, Vaccine* ajpi'iFt bacterial Med jVfmx*brpf 46:267.
Expert Committee on Brucdlosli. 19S6. ftepotft, Technical ftepon Series Nn. 740. (. ■ iv;i W1 IO.
Young hi. 1^95. An overview of human brucellosis. d in hiitei Dis. 21:2R3.

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 Mycobacterium I: Tuberculosis
Mycobacteria art slender rods that sometimes
iltOw branchiilg filamentous fort]is resembling
fungal mycelium. In liquid cultures they form a
mold-like pellicle. Hence the name 'mycobacteria’,
meaning fungus* tike bacteria. They do not stair
readily, but once stained, resist decolourlsation with
dilute mineral acids. Mycobacteria are therefore
called 'acid fast bacilli’ or AFB. They are aerobic,
nor motile, noncapsulated and nonsporing. Growth
is generally slow. T h e genus includes obligate
parasites, opportunistic pathogens and saprophytes.
T h e first member o f this genus to be identified
was the lepra bacillus discovered by I Unsen in 1969.
Koch (1882) isolated the mammalian tubercle
bacillus and proved its causative- role in tuberculosis
by satisfying Koch's postulates. Tuberculosis in
humans was subsequendy shown to be caused by
two types of the bacillus---the human and bovine
types, designated jV/ymhaerrrfijm tirbemiJosf? and
A /, ton's, respectively. The term M . tuberculosis
complex includes, besides the human and boviite types,
two other mammalian types also: M . zfricznum
causing human tuberculosis in tropical Africa and
possessing properties intermediate hetween human
and bovine Lvpe&; and AT microti (die vole bacillus
pathogenic for votes and other small mammals but
not for humans).
The second human pathogenic mycobacterium
is the Eepra bacillus causing leprosy. Though this
was the mycobacterium first described, it is the least
understood because it has not been possible to
convincingly culture it in vitro so far.
The third group o f mycobacterium is a mixed
group o f isolates from diverse sources; from birds,
cold blooded and warm blooded animals, from skin
ulcers, and from soil, water and other environmental
sources. T h e y were in itia lly called atyp ical
mycabateriii and hmadly categorised into four
categories: photochromogens, scotochromogens,
non-photochromogens and rapid growers, baaed on
their growth rates and pigmentation. They arc
opportunistic pathogens and can lead to many types
o f diseases; they arc described in Chapter 40.
Saprophytic mycobacteria were isolated from a
number o f sources. These included M l hutyricum
from butter, M . phki from grass,. M , stfwcoris from
dung and A £ smegmafj'j from smegma. (Contrary
to common belief, M . smegmatis is seldom found
in smegmaT though other rapidly grow ing
mycobacteria occur fVequenly there, contaminating
urine cultures).
Mr tuberculosis is a straight or
sliglidy curved iodhabout 3 pm x 0,3 pm, occurring
singlv, in pairs or as small clumps. The size depends
on conditions ofgrowth, and long filamentous., cluh
shaped and branching forms may be sometimes seen.
AJ. bavin is usually straiglitcr, shorter and stouter.
Tubercle bacilli have been described as Gram
positive, though strictly speaking this is not correct,
as after staining w ith basic dyes they resist
dccolourisation by alcohol even w ith o u t the
mordanting effect o f iodine. W hen stained with
carbol fuchrin by the Zichl-Neelscn method or by
fluorescent dyes (auramlne O , rhodamine), they
resist tfocofourisation by 20 per cent sulphuric acid
and absolute alcohol for 10 minutes (acid and
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352 i T e ^ lH o k
ale oho] fast). A t id fastness has been ascribed
variously to ihe presence in [he bacillus o f an
unsaponifiahlc wax (mycoioic acid) or to a
semi perm cable membrane around the cell. It is
related to the integrity o f the tell and appears Eu be
a property of the lipid-rich waxy cell wall. S ib lin g
maybe uniform or granular, Beaded or barred Forms
are frequently seen in M . ruheirulofifs, bur M . bo lt-,
stains more uniformly.
Electron micrographs o f thin sections show that
the thick cell wall is Composed of three layers
cn clo-in g a trila m in a r plasma membrane.
Spheroplasls are formed when jfrown in the
present of lysozyme. L-forms are also seen.
C u lt u r a l c h a ra c te ris tic s : The bacilli grow
slowly, the generation tune in vitro being 14-13
hours. Colonies appear in about two weeIts and mav
sometimes- take up to eight weeks- O ptim um
temperature is 37 VC and growth docs nor occur
below 25 °C or above 40 ^C. Optimum pH is 6,4­
7.0. A 1. tuherailfMiy an obligate aembe, while
M bavin is Ttiiernaeroplulic on primary' isolation,,
becoming aerobic on subculture. A/, ntbcrcu/osis
grows lliXUi lanily m culture as compared to M bin-in
which grows sparsely. They are therefore termed
ciJrpuiJO and dysgoNJC rcspccrively. The addition of
0 .5 ^ glycerol improves the growth o f AJ.
IuL>erailo$is, hut lias no effect on or may even
impair the growth of AJ. bovis. Sodium pyruvate
helps the growth o f both types. Human tubercle
bacilli do not grow in presence o f P-nirtobenzoic
acid, unlike other slow growing nonchromogens.
Tuhercle bacilli do not have exacting growth
requirements but are highly susceptible even to
traces o f toxic substances Like fatty acids in culture
media The to* u icy is neutral'.sed by serum albnmin
or charcoal. Koch originally grew the bacillus on
hear coagulated bovine serum. Severs) media, both
solid and liquid have been deseribed for the
cultivation o f tubercle bacilli. T h e solid media
contain egg (Lowenste in-Jensen, Petragnm i,
Dorset), blood Harsh is), scram (Loeffler) or potato
(Pawlowsky). T h e solid medium most w idely
dI M c& b io 'o g y *
employed for routine culture i> Lowcnstfirv-Jcnsen
i.l.ll medium without starch, as recommended by
the International Union Against Tuberculosis
(IU A T ). This consi-its of coagulated hens’ egg,
mineral salt solution, asparagine and malachite
green, the Last acting as a selective agent inhibiting
other bacteria. Among the several liquid media
described, Dubos'HMiddlebrouk’s, Proskauer a n d
Beck's, Sula’s and Sautorfs media are the more
common. Liquid media are not generally employed
for rtjut'ne cultivation, but are used for scnsirivity
testing, chemical analyses and preparation o f
an1:grns a n d vaccines.
O n solid media, M , tuberculosis form^ dry,
rough, raised, irregular colonies with a wrinkled
surface. They are creamy wh'i re, bccom I ng yellowish
or buff coloured on further incubation. They are
tenacious and not easily emulsified. M , bovis
colonies, in comparison are flat, smooth, moist,
white and break up easily when touched.
In liquid media without dispersing agents rhe
growth begins at the bottom, creeps up the sides
and forms a prominent surface ye Hide which may
extend along rhe sides above the medium. Ditfuse
growth is obtained lit Dubes’ medium containing
Lveen-fH) (sorbitan monooleate). Virulent strii'ins
tend to form long serpentine cords in liquid media,
while avirulent strains grow in a more dispersed
manner. The cord factor itself is not a virulence
factor as it is present in some aviruEent strain.'; as
well.
R e w ta n o c Mycnbarteri n are not specially heat
resistant, being killed at 60 r-'C. in 15—70 minutes.
Survival i- influenced by the material in which
the bacteria ire present. Cultures may be lulled by
exposure to direct sunlight for two hours, but bacilli
in sputum mayrremain alive for 2 0 -3 0 hours. Bacilli
in droplet nuclei may ret,tin viability for 8—10 days
under Suitable conditions. Cultures remain viable
at room temperature for 6—S months and may be
stored for up to two years at -2 0 "C.
Tubercle bacilli are relatively resistant to
chemical disinfectants, surviving exposure to 5%
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phenol, 15% sulphuric acid, 3% nitric acid, 5%
v^alir acid and 4% sodium hydroxide, They art;
sensi rive to formaldehyde and jd-urcraldehydc. 'Fhcy
are destroyed by tincture- of i(Kline- in five minutes-
and by 80% ethanol in 2-10 minutes- Ethanol is a
suitable disinfectant for skin, gloves and clinical
thermometers.
Several biochemical tests have been described
for the identification o f mycobacterial species,
Human tubercle bacilli form niacin
when jfruwn on an egg medium. When L0%
cyanogen bromide and 4% aniline in % % ethanol
am added to a suspension o f the culture, a canary
Yellow colour indicates n positive reaction, The test
is positive with human type and negative with
bovine type o f bacilli. T h e Test is useful in
identifying human
mycobacterium is positive, except for M , simaltc:
and a few strains o f M c h d o n d i
with atypical mytohaeteria.The bacilli are grown
in ;L medium containing (1.001 M rripotassium
phcnolphchalcin. disulphare. 2 N. N a O H is added
drop by drop to the culture A pink colour indicates
j positive reaction.
Virulent strains o f tubercle
hueiHi are able to bind neutral red in. alkaline buffer
solution, while lavirulent strains are unable to do
so.
These help in
d ifferen tiatin g tubercle bacilli from atypical
mycobacteria and provide an indication o f the
m : [ih - itL v itv o f i I lc S I i j l t i I i5 L s c irL ia y .L il
mycobacterial strains are strongly catalase positive,
while tubercle bacilli are only weakly positive in
comparison. On the other hand, tubercle bacilli
are peroxidase positive, hut not atypical
mycobacteria. Catalase and peroxidase activities are
lost wlien tubercle bacilli become I N i l resistant.
Catalase negative strains o f tubercle bacilli ire not
virulent for guinea pigs.
A mixture of equal volumes o f 30 vol. Ed,0,
and 0,2% catechol in distilled water is added to
5 ml o f the test culture and allowed to stand for
few minurcs. Effervescence in d icates catalase
production and browning indicates peroxidase
activity.
T h e ability h> split amide* has
heen used to differentiate mycobacteria- A useful
pattern is provided by testing five amides, viz,,
acetamide, lie-naamide, carbamide, nicotinamide and
pyrazinamide. A0.UQ165 M solution o f the amide
is incubated with rhe bacillary suspension at 37 "Xl
and 0 .1 m l of M n S O +.4 H ,0 , 1 .0 ml o-f phenol
solution and 0.5 ml o f hypochlorite solution are
added. The tubes arc placed In boiling water for
20 mi notes. A blue colour indicates a positive test.
This is positive with
M , tuberculosis and negative with M . i'>ovis.
strains as no other M any antigens have
heen identified in mycobacteria. Group specificity
is due to polysaccharide and type specificity to
This test is positive Only protein antigens. Following infection by tubercle
bacilli, delayed hypersensitive Is developed to the
hacillary protein (tuberculin). Tuberculins from
human, bovine and murine bacilli appear to be
indistinguishable. Some degree o f antigenic
relationship exists between tubercle bacilli and
atypical mycobacteria, as shown by weak cross
re-avti on$ In skin testing with different tuberculins.
There is also some antigenic relationship between
lepra and tuliercje bacilli.
By various serological tests ir has been
established tlu t AJ. rubercu/osis strains arc
snfigcnically homogeneous and very similar to
M o s t J tip ic a l X T hovf.9 .and M row rrjfr, hi:T rilsrirter from othftr
species. .Antibodies against polysaccharide, protein
and phosphatide antigens of tubercle bacilli haw;
been demonstrated in sera o f patten is, but they have
not been found useful in diagnosis or relevant in
Immunity-
M a n y mycobactcriophagcs
have been isolated from soil, water and other
environmental sources a* well as from lysogenic
Strain*. Many mycobaeterij infected with temperate
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pha^ii arc not truly lysogenic. Instead of being
integrated with the bacterial chromosome, the
phage genome appears tree, like a plasmid. This is
called pscudolytQigBny.
Tubercle bacillL have been classified into four
phage types— A , B, C , and a type intermediate
between A and B, and therefore designated I (for
’■intermediate'). Phage type A is the com i nones t and
is present worldwide. Type B is common in Europe
and N. America. Type C is seen rarely. Type 1 is
common in India and neighbouring cuointrieh.
Phage 33 D isolated from an environmental
mycobacterium lyses all variants o f M . tuberculosis
but not B C G .
AJ, fu&erru/asrs Is divisible into
two types by means o f bacceriocins produced by
rapidly growing mycobacteria.
D N A fin g erp rin tin g
provides a method for differentiating between
strains o f tubercle bacilli. Restriction endonulea&c
treatment yields nucleic acid fragments o f varying
lengths, die pattern* o f which arc strain specific.
Thin ‘rCHtncrion fragment length polymorphism1
(K F L P ) enables strain typing for epidemiological
purposes. Fingerprinting can also be done with a
chromosomal insertion sequence, IS 6110, which
is present in most strains o f tubercle bacilli.
A h the entire genome o f tubercle hacillus has
been sequenced, more molecular typing methods
may become available.
M , tuberculosis causes natural
infection in humans, other primates, dogs and some
other an imain which have close contact w ith
humans. Experimentally it is highly infectious for
guinea pigs and hamsters, hut virtually
nonpathogenic for rabbits, cats, goats, bovines and
fowl. M ice are moderately susceptible and develop
progressive infection following ititraperiruneal,
intravenous or intracerebral inoculation. Variation
in virulence among strains is frequent. Isolates, from
cutaneous and urogenital tuberculosis are often of
low virulence for experimental animals.
M . bo vis is more pathogenic for animals. Ic
produces tuberculosis in cattle, humans, other
primates, carnivores including dogs and cats,
badgers, swine, parrots and some birds o f prey.
Experimentally it is highly pathogenic for guinea
pigs and calves, moderately pathogenic for dogs,
cats, horses and rats, and nonpathogemc for fowl.
B C G , the tuberculous vaccine, is an attenuated strain
o f A f bonis;
Strains o f tubercle bacilli isolated front parts of
Africa, that show properties intermediate between
human and bovine rypes have been called African
^rainsf or M l a/fjeanum, The name 'Asian type'has
been given to strains o f tubercle bacilli originally
isolated from south India, which are o f low virulence
tor guinea pigs, susceptible to hydrogen peroxide,
isomazid sensitive and usually o f phage type L Such
strains have also been isolated from some other
Asian countries and from Asian expatriates abroad.
Though species names art sometimes used for
different varieties o f tubercle bacilli infecting
humans, they are not considered independent
entities, but only variants of die A I. fwhCK’Lr/osjs
complex.
M. nuenrti is a natural pathogen of voles. It
does not cause natural infection in humans, but can
cause ulcers after experimental infection, Bccsilsc
ic is antigen ically identical with human tubercle
bacilli, it was tried as a substitute for BCG for
human vaccination. It was given up because of
serious local reactions.
The source o f infection is usually
an open case o f pulmonary tuberculosis. Ir is
estimated that an open case of tuberculosis in India
may infect on an average some 25 contacts before
death or cure. Other forms o f tuberculosis are of
much less importance in public health. The mode
o f infection is by direct inhalation o f aerosolized
bacilli Contained in droplet nuclei o f expectorated
sputum. Coughing, sneezing and speaking release
numerous droplets— as many ah 3000 infectious
nuclei per cough. Dried bacilli in dust arc much
less infectious. Spread occurs most often among
household or other close and prolonged contacts
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Hidden page
Instead there ls w idefpH u lJutani(U [ian o f lesions
in the lungs and other organs.
Tuberculosis is an ancient
disease. Evidence u f spinal tulhrrculosis is present
in some Egyptian mummies. Tubercle bacillus
l)N A has been detected bv molecular in a
mummy dated citcn 1550-1060 BC, Tuberculosis
has been for many centuries the most important ot
human infections, in its global prevalence,
devastating morbidity and massive mortality. It has
been called die ‘white plague' and Jthe captain uf
all the men o f death',
][ is estimated that a third o f the world's
population, about two billion people are infected
with tubercle bacilli. Even1near between eight and
nine million new cases of tuberculosis appear and
three mill inn persons die from the disease. The
large majority'o f the cases and deaths arc from tbe
poor nations. India is one of the worst affected
countries. M ore than 40 per cent of the population
are infected and some 15 mi hi on suffer from
tuberculosis in the country, o f whom ewer three
million are highly infectious open cases. H alf a
mill ion people die from the disease every year in
India— one eveiy minute.
Poverty .md tuberculosis go together. W ith
improvements in standards of living, tuberculosis
had declined rapidly Lit the affluent nations, tftough
it continued uilibatcd in the poor countries. But
w ith [he progress o f the A ID S pandemic,
tuberculosis hetarnv a problem tor rhe rich nations
ulso, wi rh cases a nd even outbreaks a mong the JIIV
infected. A close relationship has emerged between
tuberculosis and H IV. N ot only does H IV infection
reactivate a blent tuberculosis infection, hut it also
makes the disease more serious and renders
treatment ineffective. Tuberculosis in torn may
h u m the development o f H IV infection into active
disease, A third complication that has made the
situ.Lriot] [lion; grave is the emergence and spread
of multifile drug resistance among tubercle bacilli.
So serious is rhe glob.il Threat o f tuberculosis
com bined w ith m u ltid m g resistance and
concomitant H IV infection that the World Health
Organic it in n lei 1W 3 took the unprecedented step
o f declaring this disease a global emergency.
Hum;-.i- Infection w ith Af. bcnii used to be
common worldwide. In many advanced countries,
as in the U K it has been almost eliminated by
regular tuberculin testing ot herds amt -l.mgtikT of
affected cattle. T h e infection spreads between
animals through aerosolised bacilli in moist cougfc
sprays. A few Infected cows shed the bacilli in milk,
which is infectious in humans when consumed raw.
The primary infection, mostly irt children would
occur iti the cervical and mesenteric lymph nodes,
fmm where it could spread to btine and joints and
other Cxtr&pulmunfiiy sites. Human infection with
Mh bovij could be prevented by drinking onlv
pasteurised or heated m ilk. Person-to-person
transmission o f of Af. bona is very rare.
Laboratory d mg nos is?
o f tuberculosis may be cstahl ished by dt monstrating
th e b acillu s in the Lesion b y m icro sco p y , iso la tin g
ir in culture or by transmitting the infection to
experim ental anim als. D em o nstration of
hypersensitivity to ruberculoptotcin maybe helpful
m some cases. Molecular diagnostic methods have
also been introduced.
T h e specimen tested is the sputum- Bacillary
shedding in sputum is abundant in cases with
cueation, but relatively scanty in organised lesions
tint do ii.ut communicate with airways, Sputum is
best collected in the morning before any meal. It
sputum is scanty, a 24-hour sample may be tested,
bputum sampling on three days Increases chances
o f detection. W here sputum in not available,
laryngeal swabs nr bronchial washings msv be
collected. In small children who tend to swallow
the sputum, gastric lavage carl be examined.
Direct nr concentration smears o f
sputum are examined. Sputum microscopy is the
most reliable singlc method in diagnosis and control
o f tuberCuloHy. MeW slides should l»e used for
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smears and tliey should not be reused, as acid fast
had Hi may nut always he removed from slides hy
cleaning. Smears should be prepared from the thick
purulent part of the sputum. Smears are dried, heat
Iked and stained by the Zichl-NccEsrn technique.
The smear is covered with strong cartel fuchsin
and gently heutEd to steaming for 5 -7 minutes,
without letting the stain boil and become dry. The
slide is then washed with water and decolourised
with 20% sulphuric acid till no more stain comes
o ff and then with 95% ethanol for two minutes.
Dccolourisation may be carried out as a single step
with acid alcohol (3% H O in 95% ethanol). After
washing, the smear is eoumerstained with Loefilers
methylene hlue, 1% picric acid or 0.2% malachite-
green far (me minute. Under the till immersion
objective, acid fast bacilli are seen as bright red
rods while the background is blue, yellow or green
depc nd i ng on the ctum terrain used- A t least 1(1,000
acid fast bacilli should be present per ml o f sputum
fbrfhem tin he readily demonstrable in direct smeam.
A negative report should not be given till at least
300 fields have been exsmined, taking about 10
minutes. A positive report can be given only if two
or more typical bacilli have been seen. Smears are
graded depending on the number o f bacilli seen:
(Tabic 39.1)
W hen several smears arc to be examined dailv. Jp
it is more convenient Co use fluorescent microscopy.
Smears are stained with auraminc phenol or
0 300 F AFB not seen
1-2 300 F [Joubtful,
repeat smear
1- 9 100 F ]+
1-9 I OF J4
1- 9 IF 3+
10 or more IF J neutralised with N /1 0 HC1 and used for smear,
NB : F-oJ immersion field
auraminc rhodamiue fluorescent dyes and when
examined lukLet ultraviolet illumination, the bacilli
w ill appear is brig ht rods against a dark
background. Because o f the contras i, the bacilli can
he siecn ever under the high dry objective, enabling
large areas of the smear to be screened rapidly.
Microscopic demonstration o f acid fast bacilli
provides only presumptive evidence o f tuberculous
infection, as even saprophytic mycobacteria may
present a sim ilar appearance. H ow ever most
saprophytic species srain un ifo rm ly w ith o u t
appearing barred or beaded and arc usually only
acid fast without being alcohol fast. As saprophytic
mycobacteria may be present in tap water, rubber
tubes, cork or hark, they can gel into- clinical
materials unless- scrupulous care is takEn in their
collection. Saprophytic bacilli are not ordinarily
present in respiratory secretions, bur they may be a
problem with gastric aspirates, feces and urogenital
specimens.
Several methods
have been described for the homogenisation and
concentration o f sputum and other specimens. They
can be classified as methods useful for microscopy
only and those useful for culture and animal
inoculation as well. To the former group belong
treatment with antiformin, sodium carbonate or
hypochlorite, detergents like tergitol, flotation
methods using hydrocarbons, and [he autoclave
method. These methods kill the bacilli without
altering their morphology or staining reaction.
Concentration method- [hat do not kill the baalli
and so can be used lor culture and. animal inoculation
InehLilr- rhe following:
T h is simple m ethod is
widely used. Sputum is incubated with an equal
volume o f 4% sodium hydroxide solution aE 37
with frequent shaking till it becomes dear, on an
average for 2U minutes. It is then centrifuged at
30(10 rpm for 20 m inutes and the sediment
culture and animal inoculation. Exciseive cjrpOiiii'C
to alkali is deleterious and should be avoided.
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 < Texloook qj Microbiology ►
A -■i inp]cr m ethod has been described
elim inating ccntrifugariim and neutral :.-at ion.
Sputum is treated w ith ait approximately equal
volum e o I a sterile locution containing 2 0 g
cetrimonium bromide and 40 g of N a O H per
!: tnc o f d i-rilled water. The contents are mi xed w: i h
a co[too swab and let stand for five minutes. About
0.2 rr1 of the inoculum i- smeared firmly with the
swab over the entire surface of acid buffered medium
(IU A T-LJ medium with 20.5 g KH^FO , per litre).
The same swab is used for inuculufitig a seen: id
slope after stirring the contents ag.iin. The results
arc as good as with Fetroff's method.
Instead o f alkali, hom ogenisation can be
achieved hv treatment with diiutc acids (6%
sulphuric acid, 3% hydrochloric acid or 5% oxalic
acid), N acetyl cysteine with N a O H , pancrealin,
desogen, zephiran and cetrimide. Flocculation
methods have also been descrilieri.
C u lt u r e Culture is a very sensitive diagnostic
technique tor tubercle bacilli, detecting as few as
10 to 100 bacilli per ml. The concentrated material
is inoculated onto at least two bottles o f IU A T -L j
medium. I f the specimen is pot-] Live by microscopy,
a direct drug sensitivity test may also he set up.
Cultures arc examined for growth after incubation
at 21 eC for four days (fn r rapid grow ing
mycobacteria, fungi and contaminant bacteria) and
at least twice weekly thereafter, A negative report
is given if no growth occurs after 3-12 weeks. Any
growth seen is smeared and tested by £ N stain-ng.
For routine purposes, a alow grow ing,
nonpiginemed, niacin positive acid fast k n ill.i- is
taken as AJ. njhejxufoxj'ff. C onfirm ation is by
detailed b^KhemKal studies. When the isolate is
niacin tteg^tvre, a battery o f tests may be needed for
identification, includir>g growth at 25 °C and 45 JC,
animal pathogenicity and biochemical tests (Tables
29.2, 59,5).
T h e use ol liquid media w i:h n id:■^i-»i^f-ic
growth detection (such as b A C T tC -4 6 0 ) and the
identification ofiholatcs by nucleic add probes have
simplified culture methods greatly and enabled
results to be given in 2-2 weeks. But these are
available only in advanced laboratnrics-
S e m id v ity tests: As. drug resistance r- an
important problem in Cuberculosifi, it is deniable
to have sensitivity o f isolates tested as an aid to
treatment. Sensitivity ic-r> for tubercle bacilli are
ma:nly o f three types. T h e first is the absolute
concentration method in winch a number o f media
containing serial concentrations o f the drugs arc
inoculated and the m in im u m in h ib ito ry
concentrations calculated. T h e second is the
res I'-.tance rat i o method in wb i ch two sets o f media
containing graded concentrations o f the drugs are
inoculated, one set with the test strain and the other
with a standard strain o f known sensitivity, The
third is the proportion method which indicates the
average sensi 1ivi tv of the strai n, tak i ng i nto account
[he fact that any population will contain cells with
vaiving degrees o f sensitivity In a drug.
A n im a l in o c u la tio n : T h e concentrated
material i- 1 uoculated intramuscularly into the thigh
o f two healthy guinea pigs about 12 weeks old,
Subcuraucous insulation is not recommended as
it leads to a local ulcer which m.iv he infectious.
The annuals arc weighed before inoculation and
at -u rervaJs thereafter. Progressive loss o f weight is
an indication n| infection. Infected animals show a
positive tuberculin sk:n reaction. One animal is
killed Lifter four weeks and autnpsled. It it shows
no evidence o f tuberculosis, the other is autopried
after fight weeks.
A t auropsv a positive animal w ill show a
caseous lesion at the site of inoculation.
Sometimes the local lesion may contain pus
under tension, which may spurt on incision. The
draining and internal lymph nodes are enlarged
and caseous. T h e spleen is enlarged, with
irregular necrotic area--, Tubercles are seen in
the peritoneum and sometimes in the lung, but
the kidneys arc unaffected. The autopsy lesions
have to be confirmed as tuberculous by acid fast
........... of smears, to exclude Y. patudotuberculosis
am! other haute rial infections which may resemble
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 G row th i m I.-J medium

Rapid growth [Whin 7 days) Stow growth

Growth on \1acConkci"
A n l Euphilasc rest
N' ill
i.iL

- +
M. fbrtuitum complex Tellmirc reduction Type of growth M. tuberculosis

+ -
M. M , pWtT figment Scanty, smooth,
flat jotontei

Rabbit pathogenic ire

In light In dark No pigment

Group 1 Gruu]i IL Group HI

PhorochromogL-u ScoTochfomqgje Nonchromogen

— +
EJ.C.G. M . hm is

the lesions o f tuberculosis), but w ill be smear
negative.
Guinea pig inoculation, once so commonly used,
is now seldom resorted no because it is cumbersome,
costly ami less sensitive than culture, particularly
with catalase negative, IN TI resistant strains isolated
from -south India.
Folym erase chain
reaction (P C R ) and ligasc chain reaction {L C R )
are used as diagnostic techniques. Transcription
medi act’d amplification, targeting ribosnimd RN A
has been introduced as an im provement on LJC R
ha-sed D N A amplification, The use o f R t’LP and
lb fingerprinting For epidemiological typing o f
strains has been referred to. Demonstration o f
miitarioo in specific drug sensitivity gtnes is a useful
indicator o f drug resistance. Such tests for
rifampiem resistance ate already available.
Serological tests arc not
useful in diagnosis, though antibodies ro many
baeLUary antigens have been demonstrated in the
sera o f patients. D etectio n o f antibody to
mycobacterial Upo-arabinom annan has been
reported to be of sonne value.

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 E-Curtun(ciassical) ■+
Asian type ■* t Aerobic
African type variable
Vole +{- variable
Bovine - -
" T O E-Thiophene-2 Carboxylic acid hydraddrHmgd)
D em onstration of’ hypersensitivity to
tubcrculuprote i.n (tuberculin tea-ti cig^) is a jtandarJ
procedure. Its scope and limitations are discussed
below.
Microscopy; culture and occasion ally animal
inoculation are used fnrdiagnosis of exTrapulimnnarv
tuberculosis also, though it is difficult to get
conclusive results as rbc bacilli arc present in far
tower numbers in these lesion? than in pulmonary
disease. Ci>K from tuberculous meningitis often
develops ;l spider w eb dot on sta n d in g , examination
o f which maybe more successful than o f the fluid.
I’hc Lise o f P C R and D N A probes may help detect
tile bacdli speedily and more often.
Bone marrow and liver biopsy specimens from
m iliary tuberculosis and blood from those wirh.
H IV coinfection are useful for culture. PUs from
tuberculous abscesses often yields positive results
in smear and cultutC-
Plcural effusion mid oilier exudates may be
collected with citrate to prevent coagulation. I f free
from other bacteria* they may he used for culture
after centrifugation- I f other bacteria are present,
prior concentration is necessary.
Urinary excretion of bacilli in renal tuberculosis
in intermittent. Hence it is advisable ro test 3-6
m orning samples o f urine. Each sample is
Centrifuged for .3000 rpm for .30 minutes and the
sediment used for culture after concentration.
Infection w itb
tubercle bacillus induces cell mediated immunity
Aerobic 1- A.B.C
- l
Micrvaerophilie - A
Mkroaemphilic - -j
MiaoteropbiBc - A
which is manifested as delayed hypersensitivity
{allergy) and resistance to infection (immunity). The
resultant of these two processes determines the
course Lit' rhe infection.
The response o f a tuberculous animal to
reinfection was originally described by Knch,
Subcutaneous injection o f virulent tubercle bacillus
m a normal guinea nig produces no immediate
response, bur after 10—14 days a nodule develops at
the sitn.% which breaks down to form an ulcer that
persists till the anim al dies ot progressive
tuberculosis. T h e draining lym ph nodes are
enlarged and caseous. I f on the other hand, virulent
tubercle hat-ilkis is injected in a guinea pig which
had received a prior injection o f the tubercle bacillus
4-6 wcljL% earlier, an indurated lesion appears ar
the sire in a day or two. This undergoes necrosis in
another day or so to form a shallow ulcer that heals
rapidly wit bout involving the draining nodes or
other tissues. T h is is known as the K och
phenom enon and is a combination o f hyper­
sensitivity and immunity. The Koch phenomenon
has three component;*— a ‘local’ reaction, a *focaT
response m anifested as congestion and even
hemorrhage around the tuberculous foci in tissue*,
and a constitutional' or 'systemic' response of fever
which may sometimes He fatal-
A llergy can be induced by infection with
virulent as well as avirulent tubercle bacilli, but
nor ordinarily by injection o f killed bacilli ot
bacterial proteins. However* fo r d e m o n s tr a ti n g
allergy, live nr killed Iran Hi or tuberculoprotein can
be employed-
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Hidden page
262 * Texloook Ol Microbiology n
o f early detection and treatm ent o f Cites,
chemoprophylaxis and immunoprophylaxis.
Immunoprophylaxis is h-v iiitradermal injection
o f the live attenuated vaccine developed by Calmette
and Guerin (1921), the Bacille Calmette Guerin
or KCG. T h i- H a strain o f M. hijvix attenuated by
229 serial subcultures In a glycei:ne-bilc-potato
medium over u period o f 12 yean. Injection o f E C G
in animals induces self lim ited infection, with
multiplicalmi: .md disseiitinatiurt o f the bacillus ill
different organs aid production o f small tubercles.
W ith in a few weeks the bacilli stop multiplying
although they survive in the tissues furlong periods.
This gives rise to delayed hypersensitivity and
immunity. Following BCG vaccination, a tuberculin
negative recipient is converted to & positive reactor.
T h e im m unity may last for 10-15 years and is
similar to the immunity following natural infection,
except that it does not carry any risk o f disease due
to reactivation, as in the latter case.
Sevtral field trials have Keen conducted to assess
rhe efficacy o f the B C G vaccine. The results have
varied widely, from flO per cent protection to a total
absence o f protection. In south India, a trial
conducted ar Madanapalle showed 60 per cent
efficacy while a later large trial in Chmgalpattu
did not reveal any protection in adults, though a
15-year follow up showed some protection in young
persons. The reasons for such wide disparity are
not clear hut have been attributed to several factors
such as the differences in the prevalence and
virulence o f tubercle bacilli in various communities,
the type and potency o f vacc ines used and the
presence o f ’atypical mycobacteria* in the areas,
The E C G vaccine aroused much cridcism and
11 has been suggested that i t may regai n iits vimlencc,
though there has not been any evidence ol it so far.
The Lubeck disaster in which several children
developed fatal tuberculosis fo llo w in g oral
immunisation tamed the vaccine much notoriety'
■n the early days o f the vaccine. This was due to a
mix-up by which "ivc virulent tubercle was given
instead o f B C G . Stringent safety measures have
since been enforced in [he manufacture o f B C G
vaccine, The recogni-ed complications o f B C G
vaccine are the following:
Locti: Abscess, indolent uker, kelnid, tuberculides,
confluent lesions, lupoid lesions, lupus vulgaris,
im il E] iLajgeitient and uppuiati on-ofdnti i Ling
lymph nodes.
GeceraJ: Fever, mediasunal adenitis, erythema
nodosum, tendency to keloid form al ion after
wounding at other sires, and very rarely nonfatal
meningitis. T h e very few cases o f progressive
tuberculosis reported are believed to have been in
immunodefiL'ient subjects.
The consensus opinion is that B C G may not
protect from the risk o f tuberculosis infection, but
gr.res protection to infants and young children
against the more serious types o f the disease, such
as meningitis and disseminated tuberculosis. The
recom m endal inn therefore is that in endem ic
countries such as India, BCG vaccine be
administered to babies by mtradermal injection on
the deltoid immediately after birth, or as early as
possible rhereatter, before the age o f 12 months.
The vaoci ne need rot be administered after the age
of two years, B C G should not be given to infants
and children with active H IV disease, though it
may be given with benefit to asymptomatic H IV
pui-1t 1vest. Babies bunt to mothers wi Lh A FB pos 11ive
sputum should not be ghen B C G at birth, hut only
after a course o f preventive chemotherapy.
ECG nduces a nonspecific stimulation of the
immune system providing some protection against
lepmsy and leukeni.i- Multiple injection of BCG
has been tried as adjunctive therapy in some
malignancies. Some workers have reported that
BCG is supcr^n to PPD for tuberculin testing.
Chemoprophylaxis or preventive chemotherapy
id the administration o f antituberculous drug-,
(usually only isoniazid) to persons w ith latent
tuberculosis (asymptomatic tuberculin positive] and
a high risk o f developing active tuberculosis, or to
the uninfected exposed to high risk of infection. It
is particularly indicated in infants of mothers with
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active tuberculosis and in children living with a
case o f active DubeireuloriFi in the house. Isoniarid 5
nsg/kg daily for &-J2 months is the usual course,
Trial* have shown that this reduces the risk of
developing active disease by 90 per cent. I IIV
infected contacts cl active tuberculosis also benefit
burn this prophylaxis.
C hem otherapy has revolutionised the management
o f tuberculosis. It has been established that
Sanatorium regimens, bed rest, fresh air and rich
food t as well as operative interventions, such as
artificial pneum othorax and thnruonptasrv arc not
essential for cure it domiciliary treatment w ith
effective antituberculous drugs is given in optimal
dose and duration.
A ntituberculous drugs are o f two types,
bactericidal anti bacteristatic. O f the bactericidal
drugs, rifampicin (R) and pyrazinamide £2) arc
called sterilising drugs because they are able to
effectively hill the bacilli in the lesions. O f the other
bactericidal drugs, isoniazid (H ) is elective H>nly
against repheating bacilli and streptomycin (S) only
against extra cellular bacilli and so are not by
themselves able to sterilise rhe lesions. The
bactericidal drugs, along with cite bacteristatic drug
etbarnbutol (E) constitute the first line drugs in
antituberculous therapy, J|bc old practice o f daily
administration of drugs for two year* or SO has been
replaced by short cuurae regimens o f 6 -7 months,
which arc effective and convenient. A typical
example o f such a schedule fora new smear positive
case ia a combination o f four dreg* (H R Z E ) given
three times a week during an initial intensive phase
o f TWO m onths, follow ed by 4 “ 5 mouths ul
continuing phase with only two dreg* (H R ) three
times a week.
The major problem in chemotherapy is drug
resistance , which in tubercle bacilli is due to
mutation, with an approximate rate o f once in 10s
cell divisions. This could have been effectively
cheeked by multiple drug therapy, which was
introduced for this very purpose. Unfortunately this
was not im plem ented properly. D u e tn a
combination o f lapses in prescribing practices, drug
delivery a,ndpatient compliance, resistance has built
up in tubercle bacilli, over the years, reducing the
efficacy o f treatment
Drug resistance may be 'primary' [pretrearmem,
initial), when the patient is infected with a strain
o f tubercle bacillus which is already resistant, or
'acquired’ (secondary, post-treatment), when the
infecting strain initially sensitive becomes resistant,
usually as a result o f improper or insdequate
treatment. This is the more common type of
resistance. W hen acquired resistant strains become
increasingly common in an area, the chance o f new
patients presenting w ith prim ary resistance
increases. W hen an infecting strain acquires
resistance to one drug, the chance o f its becoming
resistant to other drugs increase*, unless the
treatment schedule contains an adequate number
o f effective dni££. A t present the spectrum o f sir jit I s
prevalent in the com my nitv cover* resistance to all
available antituberculous drugs.
A very serious consequence o f unchecked drug
resistance has been the emergence and spread of
m ultidrug resistant tuberculosis (M D E U T B ).
Though the term multidrug resistance means only
resistance to two or more drugs, in the context of
tuberculosis, it specifically refers to resistance to
rifampicin .md isunifizid, with or without resistance
to -Line or more other drugs. This is because R and
H form, the sheet anchor o f sh ort-term
chemotherapy and any strain resistant to both three
drugs is unlikely to respond to treatment.
M D R -T B is a global problem, menacing the
poor and the rich nation* alike. It may he primary
or acquired- Its presence in those with concomitant
H IV infection maltes it more dangerous. W hen first
line drugs become ineffective, second line drugs
have to Sic tried. T^arge numbera ut old and new
drugs are being used - qumolonc*, aminoglycosides,
macnolidcs, para amino salicylic acid, thiacctazone,
cycloserine, iiapreomyein and others. They are
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Hidden page
 Mycobacterium II: Non-Tuberculous
Mycobacteria (NTM)
M ycobacteria other than man mall an tubercle
bacilli, which imay occasionally cnee human disease
resembling tuberculosis, have been vailed 'atypical',
'anonymous' or 'unclassified' mycobacteria. Tile
names ‘environm e ntal' or O pportunistic
mycobacteria1 are better suited as their natural
habitat appears to be soil or water and they cause
opportunistic infections in human beings.The name
'uontuberculous mycobacteria’ (N T M ) has gained
wide acceptance in recent yearn. They hare also
been called ‘paratuberclc’, Ltuberculoid7 and
'M O T T ' [mycobacteria other than tubercle) bacilli.
They are distinct iro n the saprophytic mycobacteria
such as .1/. phlei which are incapable o f infecting
human beings or animals. W hile human infection
with them is common in some areas, disease is rare.
They are unable ro cause progressive disease when
injected into guinea pigs.
N on -tuberculous mycobacteria have been
clarified into four groups hy Rouyon [1959) based
cm pigment pnoducrlon and rate of growth: Group
1 photochromogens, Group El scoroehromogcns,
Group I I I nonphotochromogens and Croup IV
rapid growers. Though other methods of
classification have bee it described, Runyon's
classification lias found Universal acceptance.
Species identification depends on several additional
characteristics [Table 4 0 .1).
These strains
form colonics that produce no pigment in the dark
bur when [lie young culture is exposed to light for
one hour in die presence o f air, and reincubatcd for
2 4 -2 3 hours, a yellow orange pigment appears.
They are slow growing,, [hough growth ls faster
than that » f mheitle bacilli. Tin- important species
in rhi? group are M . Jqui.^zffil, Af. marimmi and
M . simsae.
Mr kxasasii causes chronic pulmonary disease
resembling tuberculosis. usUllly affecting the upper
lobes, w ith cavity form ation and scarring,
M kanszsii has beer isolated from tap water
samples around the world and this ls believed to
be [he main reason and source of infection. It is the
second most common N T M seen in lung diseases
alter A/, avium complex.
M . jiiarifliirn which causes a warty skin Lesion
("swimming pool or fish tank granuloma"), closely
resembles Af. kansasit but can be differentiated by
its poor growth at 37 GC , negative nitratasc, positive
pyr.trin am idr- hydrolase and T.-JiLcosidase activities.
Several piiotochromogemc mycobacteria were
isolated in L964 from monkeys caponed from India.
They have been classified into two species: niacin
positive A f siriijae and niacin negative Af. avai^um.
T h e y hive subsequently been associated w ith
pulmonary disease in human beings.
These strains
form pigmented colonies [yelW'-orange-red) even
in the dark. They are widely disrribured in the
environment and sometimes contaminate cultures
o f tubercle bacilli. A"f. scm fiibreiini mav cause
scrofula (cervical ademtis) in children.
M. gunJonae, often found in tap water (hence
called 'the tap w afer scotnchrnmngen'}, is a
common contaminant in clinical specimens and a
rare cause o f pulmonary disease. U differs From AJ,
scrofu^iceum in failin g to hydrolyse urea,
nicotinamide and pynzinamide.
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Growth in 7 days ■» 4 4 - 4

Growth at 25 °C * i 4 4 4

<JimiTh at .17 + 4 4 * 4 4 4

Growth it 45 °C - 4 4

Pigment in dark * #
Pigment in light + - 4

Urease 4 + 4 +
Niacin
Nitrate reduction + 4 +

Mr szulgii is scorochrotnogenic when grown
at 37 °C and photochromogenic at 25 JC.
These
strains do not form pigment even on rvpoRurc light.
Colonies may resemble those o f tubercle bacilli;
The medical iv important species are M. avium,
Afr intnaed/tiJitrc, M , jrcjiopj and the skin pathogen
M . Lt/eerans.
M avium, which causes namral tuberculosis in
birds and lymphadenopathy in pigs is one of
the most common opportunist human pathogens.
Af, in IrAccUuhirc is eon imu idv known ns the 'Rattcy
bacillus' Iwcause it was first identified as a human
pathogen at ihe E n tity State: H o sp ital for
Tuberculosis, Georgia, USA.
jVh avTum and M , intracellulafe are so closely
similar that they have been considered as one
group, the M . avium complex (M A C ), They cause
lymph adenopathy, pulmonary legions or dissemi­
nated disease, particularly in A ID S pacienii. They
are related to the animal pathogens M. parumbcr
culosis and Ai. iepnsernuricjn?.
Af. nraJmijense which may cause pulmonary
disrate is a slow grower, taking 8 -12 weeks to form
colonics.
A f xrnopi, originally isolated from toads, may

Distribution Tropics Temperate zone

Clinical course Chronic progressive ulcer Self'limited ulcer
Bacilli m ulcer Abundant Scanty
Rnre of groin h Slower; 1 8 weeks I'aster, 1-2 weeks
(Jrowrh jr 25 *C - +
Growrh at 37 JC - 4
Culture film Bacilli in cord* No cold formation
Pigment in Lighr - +
.Miiukc iuutpad Lefion Edema, rarely ulcer Marked inflammation—purulent
ulcer

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occasionally cause chronic lung disease in human
beings. M. xtiTQfi und AT. itviuitt t*fe thermophiles,
capable of growth at 45 QC.
Though usually classified as a nonpboto-
chrom ogcn, Af. xenopi may form seoto-
chromogenic yellow colonies. M . xenopi has been
Isolated from water taps, mostly hot water raps, in
hospitals. It has also been isolated from main water
supplies.
This is i
heterogeneous group of mycobacteria capable of
rapid growth, colonics appearing within seven days
o f Incubation at 37 6C or 25 DC. W ith in the group.
photochrom ogcnic, K O to eb ro m o g rric, and
nemchromogeoic species occur. Chrornogtnic rapid
growers Lire mostly saprophytes (for example, Af.
phlci). The medically important species are M ,
fortiij'tu/i] and M . chelonzc both of which can cause
chronic abscesses in human beings. Outbreaks of
abscesses following injection of vaccines and other
preparations eomammited by these mycobacteria
have been reported flfi a Humber o f ocCasicn ls . The
bacilli are (bund in the soil, and infection usually
follows some injury
Af. fomurum and M . efidonci do not produce
any pigment, Pulmonary lesion* caused by M ,
/bm dfum cannot be distinguished radiologic ally
from typical tuberculosis. No effective
chem otherapy is available. M . itnegmadf,
commonly considered as saprophyte in smegma, is
seldom seen in that location. It is a frequent isolate
from soft tissue lesion* following trauma or surgery.
Sortie nonculrivablc or poorly growing
mycobacteria identified from the blood o f AIITS
patients have been characterised by their lfi,S RNA
base sequences. "Ibey grow sparsely in some liquid
media- Examples are jVf. ^eneven.se', IV/. conilucntis
and A/, jurenncdium,
A rapid grower, M . raccae is reported to be an
i m mu nomoduS ator capable of inhibiting tissue
destroying hypersensitivity responses and
Stimulating protective im m une processes in
tuberculosis. Clinical trials of M . n e a t vaccine
as an adjuvant to chemotherapy in tuberculosis am
on.
Cutaneous lesions may occur in leprosy or
tuberculosis, cither as localised disease or as part
o f a generalised infection. In a different class are
two species o f inycohacteria, A/- tdcfrttrtS and Af.
mAnnum, which arc exclusively shin pathogens,
causing chronic ulcers and granulomatous lesions
On the skid. Systemic invasion dues not occur and
the regional lym ph glands are not involved.
Cutaneous localisation is because they multiply
optimally at skin Temperature.
This WJS originally isolated from
human skin lesions in Australia (IS4ff) hut has
subsequently been recovered from similar lesions
front Uganda (liu ru li ulcer), Congo. Nigeria.
Mexico, Malaysia and New Guinea. Ulcers are
usually seen on the legs or arms and are believed to
follow infection through minor injuries. After an
incubathm period oi a few weeks, indurated nodules
appear, which break down forming indolent ulcers
which slowly extend under the skin,
Initially smears from the edge o f the ulcer show
large clumps o f bacilli which are acid fast and
alcohol fast. Later, the imtpunorcactjvc phase sets
In and the bacilli disappear. The ulccn then heal
with disfiguring scare.
T h e bacillus grow s on I^ow custein-Jenscn
medium slowly, in 4-E weeks. The temperature
o f incubation ls critical' growth occurs between
.10 ° c and 33 °C but not at 25 °C or 37 Inocu­
lation Into the foot pad o f mice leads to edema of
die limb though ulceration is- infrequent. A toxin
is produced by A f. uicerana th a t cause*
inflammation and necrosis when injected into the
skin o f guinea pigs. This is the onty known instance
o f toxin produced by any mycobacterium species.
This is a natural pathogen o f
coldblooded animals, causing tuberaihisis in fifth
and amphibia. It may also occur as a saprophyte in
fresh or salt water, I luman infection originates from
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 N . aHinsnunt Animals, toil Pulmonary
Mi a n ftb ru m Primates Pulmonary
M avium rrrfracei/ulare Soil, seawater animals Pulmonary, aj^temk,gastrointestinal,
lymphadenitis
Mi cheknae Soil, « awater, uni mals E^mcirie heart valves,suigitTil wound, pulmonary
M . chelonae* s.s. Soil, seawater animals Cutaneous, surgical abscessus w o u n d -
pulmonary*EyElemic
M i fitllax Water, soil Pulmonary
M. iortuitum Water, soil animals Pulmonary, surpical wound cutaneous, systemic,
hew awl joint
M, huentophiluin Unknown Cutaneous, tubcutaneou*
Af kantisii Water, animals Pulmonary systemic, skin joints, lymphnodcs
M. nwhnvense Unknown Pulmonary
M. jrMnrtirrri Aquarium water, fish Cutaneous (swimming pool granuloma),
joint*
M . sciofuliceum Si nl, water, fomitea Lymphadenitis (usually cervical);
p u lm o n aT V dmemmited
AJ_ schimcidcs Unknown Pulmonary
M. srmrac primates, water Pulmonary
M . szulg-ii l fnknown Pulmonary, lymphadenitis, cutaneous,
BubcutaneoLLS bursitis
Al. uk'LTJJlS l fnknown Cutaneous
M. HlK^' Soil, water Pulmonary, epididymitis

cumriunimLred swimming pools or fish tank*. TEic
lesion, beginning .is a papule aril breaking dmvn
to form an indolent nuclear, usually follows
abrasions and there fi|>re iHicurs on [fie prominences
— elbows, knees, ankles, nose, lingers or toes, ft
was first described from Sweden under the name
"■SWirmriing p o o l g r a n u lo m a ', a n d t f ic h a p llU H w as
named M . bsdm-L (from ba/ncLt/n meaning tv-irh). !r
has since been reported from other European
countries and from North America. Its distribution
is in temperate areas in contrast to M . ulcentist
which has a tropical prevalence. El uni an infection
may occur in epidemic form. The ulcers are self-
limited and undergo spontaneous healing-
Hacifii are scanty in *m e,ir 5 from ulcers,
Growth occurs in about two weeks at 30 '"C (range
25-35 * € ) and primary cultures do not grow jit
37 *C but they do so after adaptation. Colonies arc
nonpigmented in the dirk; however, they lieeon n e
intense orange yellow to red on exposure to light,
M . n w ijiu iii is not pathogen ic for guinea pigs but
intraderm ul inoculation in r a b b i t s leads t o a
superficial granulom atous lesion- Pootpad
inoculation in mice lc,ids to a more severe lesion
than with M . ideenuu, local inflammation being
followed hy a purulent u1«r formation.
Infection w ith M . m arrnum , but not M .
uJccniJit, may cause ,1 low-grade tuberculin reaction,
A/. hitm ophihim , firEi described in 1978,
causes skin lesions. It requires Liecom for growth.
It grows at .12 *C in 2-4 weeks hut notnt 37 °C-
Tablc 40,3 shows the range o f human infections
produced hy differen t species o f atypical
mycobacteria.
As environ*
mental bacteria are widely distributed in nature,
infection with them is quite common, from soil,
wafer and air. Pfcrenn-Jo-person infection docs not
seem to occur. Infection is mainly asymptomatic,
though it may result in sensitisation, causing weak

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Hidden page
 Mycobacterium III: M. Leprae
Leprosy is j disease of great antiquity, having been
recognised from Vcdit rimes in India anil from
biblical times in the M iddle Last, lr probably
originated in the tropics and spread to the rest of
the w orld. Leprosy has alway> been held in
superstitious dread and the person suffering from
leprosy considered 'unclean and a soda! outcasts.
The lepra, bad] Llis was first observed by Hansen in
IfldEf. Though this wan the first bacterial pathogen
o f humans to be described, it remains one o f the
least understood. This is because it has not been
possible to grow the bacillun in culture media.
M . leprae is a straight or slightly
curved md, 1-K urn * O.idJ.S pin in sise, showing
considc table raorpholog Lcnl vari atio ns, Poln r bod its
and olI lct intracellular elements may be present.
Clubbed forms, lateral buds or branching may be
observed. It is Gram positive and stains more readily
than the tubercle bacillus. It is acid fast, bur less so
than tubercle bacillus. Hence 5% sulphuric acid
instead ot 50% is emphiyed for ilecnlouri Ration atler
staining with carbol tiicfoin. It is the practice to
differentiate between live and dead bacilli in stained
smears, assuming without CDndusivf proof that the
former appear solid and uniformly stained, while
the latter are fragm ented and granular. T h e
percentage o f uniformly stained bacilli in tissues
{m orphological index) provides ,t method o f
assessing the progress o f patients on chemotherapy
and is more meaningful that! the old c r ite rio n of
bacteriological iir d u (the number o f hue ill i in
tissues).
T h e bacilli are seen singly and in groups,
intraLcIliiUrly or lying free outside the cells. They
frequently .ippesir as agglo me rates, the bacilli heing
bound together hy ,1 lipid-like substance, the glia-
Fhesc masses are known as 'globi'. The parallel
rows o f bacilli in the gtobi present a "cigar bundle'
appearance, In tissue sections, the clumps o f bacilli
resemble cigarette ends. T h e gtobi appear in
Virchow's 'lepra celh' or Toatny cells' which are
large undifferentiated histiocytes.
It has not so for been possible to
cultivate Lepra bacilli either in bacteriological media
ui in tissue culture, There have been several reports
o f successful cu ltivation but none has been
confirmed. One of the best known o f such reports
(1% 5) came from the Indian Cancer Research
Centre, Bombay, where Lin acid fast bacillus was
isolated from leprosy patients, employing human
fetul spinal ganglion cell culture. This IC R C
bacillus has been adapted fo r grow th on
Lawenstcin-Jenser medium. Its relation to the lepra
bacillus is uncertain.
There have been raanv attempts to transmit
leprosy to experimental animals. However, the real
break through was rbc discovery by Shepard (1960)
that lepra bacilli could multiply in Hie firntpads of
mice kept at a low temperature (20 r C), This
observation bias been confirmed and has become
the standard procedure for experimental work with
the bacillus. Hollowingmrradcrmal inoculation into
the footpads of mice, a granuloma develop! at the
site ii l 1—f> months. If cell mediated immunity is
suppressed bv thymectomy or the administration
o f amilyitlphocrte serum, a generalised infection is
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 « Mycobacterium III- M Leprae *
produced, simulating lepromatoua leprosy. The
nine-banded armadillo {Dasypus novemcvi^ctus) is
highly susceptible to infection with lepra bacilli.
Follow ing inoculation into armad.ilIocs, a
generalised infection occurs w ith extensive
m ultiplication o f the bacilli and production of
lr-.iims typical of lepromatous leprosy. Some c\ iLJ
armsui Hoes in capfivity have been observed to be
naturally infected with a mycobacterium resembling
the lepra bad!Ins. "Natural disease' has also been
identified in chimpanzees and mangabey monkey?:
from West Africa but ir is nor known whether they
have any relevance to human infection.
In animal experiments, rhe generation time of
the lepra bacillus has beer found to be excepl innallv
long, 12-13 days on the average but may vary from
£ -4 2 days, in comparison with about 14 hours in
the case of the tubercle bacillus and ahout 2Q minutes
in the case o f coliform bacilli
Af. leprae genome has been mapped and the
genes for its major protcifi antigens cloned and
seque need­
le c-si si n nee: Lepra bacilli have hcen found ro
remain viable in a warm hum'.d environment for
9 -1 6 days and in moi ‘■t soil for 4b days. ‘Ilic y surv ivc
exposure to direct sunlight for two hours and
ultraviolet light for 30 minutes.
LEPROSY
Leprosy is a chrome granulomatous disease ° f
humans primarily involving [he skin, peripheral
nerves and nasal mucosa but capable o f after: i.ng
any tissue or organ. The disease may be classiried
into four types — Scp r o m a ta u a , t u b e r c u lo id ,
rii m orpho uy and i ode term inate (M a d rid
classification, 1953). T h e type o f disease is a
reflection o f the immune status o f the host. It in
therefore not perm anent and varies w ith
chemotherapy and alterations in host resistance.
Bacilli 'i-*diitcd from dilferent types u f leprotv do
not differ in virulence or other ptopcrties.
T h e two extreme or ‘polar’ forms o f the disease
are the lepromatous and tuberculoid types. T h e
371
lepromatous type is seen where the host resistance
is low. The bacilli are seen in large numhers or as
globi inside lepra cells or extracellular])-. This is
known as 'm u ltib aciIIary disease’ . Superficial
nodular lesions (lcpnomata) develop which consist
o f granulation tissue containing a dense collection
o f vacuolated cells in d ifferen t stages o f
development Iron: mnnonudeir cells to lepra cells.
Tbc nodules ulcerate, become secondarily infected
and cause distortion and mutilation, Bacilli invade
the mucosa o f the nose, mouth and upper respiratory
tract and are shed in latgc numbers in nasal and
oral secretions. The reticuloendothelial system, eyes-,
testes, kidneys and bones ate also involved.
Eacillemia is common. The lepromatous type lS
more infective than the other types. The prognosis
Is poor. Cell mediated immunity' is deficient and
the lepromin test ls negative m lepromatous leprosy.
O n the other hand, there in an exaggerated and
broad humoral immune response. Antibodies in
high litres are seen against mycobacterial as well
as several other antigens. A utoan tibodies ire
common. Most cases show hliologi. aJ false positive
reaction in standard serological tests fnr syphilis.
A t the other end o f the spectrum is tubexddoid
leprosy, which is seen in patients with a high degree
o f resistance. The skin lesions are few and sharply
demarcated, con-.isTin^ o f macular anesthetic
patches. Neural involvement occurs early and may
be pronounced, leading tn deformiUL'S, particularly
in the hands and feet. Bacilli are scanty in the
lesions and infectivity is minimal. This it known as
'paucihacillarv disease*. Celt mediated immunity i.£
adequate and the leprom in test is p O lith r .
Aiidmycobaeterial and autoimmune antibodies are
1, 11c. The prognosi- is good.
The term borderline or dimorphous type refers
to lesions possessing characteristic! o f both
tuberculoid and lepromatous types. It may shift ta
the lepromatous or tuberculoid port o f the spectrum
depending on chemotherapy or alterations in host
resistance.
The indeterminate type is the early unstable
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 T h e deficiency in cell mediated im m u n ity is
specific [u [lit: lepra bacillus antigens. 1 .cpromatous
patients arc not more susceptible to viruses, parasites
acid o th e r p a th o g e n s a g a in st w h ic h C M ! is
important. Tuberculin reactivity may be suppressed
in untreated LeprnmatouR patients but it becomes
positive following treatment, unlike the lepromin
response w h ich rem ains negative Le p ro m aio u a
patients have large number* o f C H S * (tup p raso rj
ly m p h o c y te s in c ir c u la t io n , w h ic h c a n be
specifically activated by the lepra bacillus antigens.
T h e lym phocytes present in skin granulomas are
alm o st e xclu sively C D S + ce lls, in co ntrast to
Tuberculoid lesion* w inch contain predom inantly
C D 4 + ce lls. I n lep ro m ato u s lepro sy, there it
extensive polyclonal li cell activation w ith large
am o u n ts o f antibodies b e in g p ro d u ced , hnth
a n ti m y c o b a c te ria l an d a u to im m u n e . T h e
album in g lo b u lin ratio is reversed, T h e antiinyco-
bactcrial antibodies are nor beneficial. O n the other
hand, they may hive an enhancing effect.
T h ere is evidence of genetic effect in the pattern
o f response to lepra bacillus infection. H L A - D R 2
is seen p re p o n d e ra n tly in perso ns w ith the
Lul^rculoid type o f reaction, while H L A - M T 1 and
H L A - D Q l a r c associated with lepromatous disease.
T h o u g h leprosy in a chronic disease, its course
may he interspersed with acute exacerbations w hich
are o f an allergic nature-Tw o types o f such reactions
occur.
Type 1 lit the 'reversal reaction or the ‘lepra
reaction ' is seen m ostly in borderline leprosy.
Lepra bacilli in tissues ± +
Lepromin test +*
Mycobacterial antibodies ± +
Lymphocytic infiltration of lesions ++ 4
Plasma cell in lymphoid tissue 4 +
o ccurring sp o ntan eo u sly or mote often d u rin g
ch em o th erap y. It is a c e ll m e d ia te d im m u n e
reaction, with an influx o f lym phocytes into lesions,
a n d a shift to tuberculoid morphology. T h e lesions
develop ervlhein.i and swelling, along w ith pain
and tenderness- A sim ilar clinical picture is seen in
the ‘d ow ngrading reaction' w hich occurs usually
in untreated or pregnant patients. H ere, biopsy o f
rhe lesions shows a shift to the lepromstcms pattern,
reflecting .1 decrease o f C M I -
T y p e 2 re a c tio n or E r y t h e m a N o d o s u m
Leprosum ( E N L ) occurs in the L L and B L types,
USUI llv a few m o n th s afte r in s t it u t io n of
ch e m o th e ra p y . C ro p s o f te n d e r, in fla m e d
su b c u ta n e o u s n o d u le s appear, w ith fever,
lympbudeuopatbv and arthralgia. T h is is an ArthuS
type response to antigens released from dead lepra
cd ls and is characterised by neutrophil infiltration
and Ig G and com plem ent deposition in the lesions.
T ill recently, the only method for studying im m unity
in leprosy was a skin test lor delayed hypersensitivity,
the lepromin test first described by M itsuda in 19 19 .
T h e o rig in a l an tig e n (Je p ro m in J w as b o ile d ,
em ulsified, lepromatous tissue rich in lepra bacilli,.
T h e response to the in t ia d e r m il in je c tio n o f
leprom in is typically biphasic, consisting o f two
separate events- The first is the early reaction of
Fe rnandez., w h ic h c o n s is ts o f e ry th e m a an d
induration developing in 2 < W 8 hours and usually
rem aining for 3 -5 davs. T h i* is analogous to the
+* +
+ -
+* +
+ -
+* +
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 374 i Texlbraa* 01 Microbiology ►
tuberculin reaction. T h is is usually poorly defined
and carries little significance. T h e second and more
meaningful is the late reaction ot M itsu iL l, starring
in 1 - 2 weeks, reaching a peak in four weeks and
gradually subsiding in rhe next few weeks. T h e
reaction consists o f an indurated skin nodule, w hich
m ay ulcerate. Histologically, there is infiltration with
lym phocytes, epithelioid cells and giant cells. T h e
M itnuda btc reaction docs not indicate pre-existing
D T I L but i s a measure o f the C M I induced by the
in je cte d le p ro m in itself. I t thus d istin g u ish e s
between persons who can m ount a C M I response
against the lepra bacillus antigens and those who
cannot.
T h e original crude M itsud a antigens extracted
from skin le-ions o f leprornatoLisi patients (integral
lepromins) were standardised on the basis o f tissue
c o n te n t. M o d e r n a n tig e n s are sta n d a rd is e d
according to their lepra bacillus content (4 jc. 10 T
lepra bacili; per m l). Standard lepromins arc be mg
It Li ■.irl..I Ltwrcj-ingly from armadillo derived lepra
b acilli ( le p r o m in -A ), replacing hum an derived
leproiflin-H .
T h e lepro m in test is not used to d iag n o se
leprosy, nor does it indicate prior contact w ith the
lepra bacillus. H e alth y persons in nonendemiL: areas
w ith no chance o f contact with the bacillus may
give a positive leprom in test. T h e test is employed
for the following purposes
1. T o classify the lesions o f leprosy patients. T h e
leprom in test is positive in tuberculoid, negative
m lepromatous and variable in dimorphous and
indeterminate types o f d^casc-
2 . T u assess the prognosi-. and response to treatment.
A positive reaction indicates good prognosis and
a negative o re bad prognosis. C o n versio n to
leprom in positivity during treatment is evidence
o f improvement.
3. To assess the resistance ol individuals to leprosy.
If is de-crable to recruit only lepromin positive
persons for w ork in lep ro saria as lep ro m in
negative persons are mote prone to develop the
disease.
4. To verify tht identity iif candiiLste lepra bacilli,
Cu ltivable acid fast bacilli clu'imed to be lepra
bacilli should give match mg results when tested
m parallel with standard lepromin.
la b o r a to r y d ia g n o s is : B a c t e r io lo g ic a l
diagnosis is easy in the lepromatnus but may be
d ifficu lt in. the tuberculoid eases. T h e diagnosis
consists o f demonstration o f acid fast bacilli in the
les^m s, For routine exam ination, specim ens are
collected from the nasal mucosa, skin lesions and
eat lobules. A blunt, narrow scalpel is introduced
into the ro se and the internal septum scraped
sufficiently to remove a piece o f m ucus membrane,
w hich is transferred to a slide and teamed out into a
uniform smear. Samples from the skin should be
obtained from the edges o f the lesion rather than
from the centre. T h e skin is pinched up tit;ht to
m inim ise bleeding and a cut about 5 mm long made
with a scalpel, deep enough to get into the infiltrated
layers. After w iping o ff blood or lym ph that may
have exuded* the scalpel blade i; turned transvenely
to scrape the sides and bottom o f the cut so as to
o b ta in a U tile tissue p u lp w h ic h Is sm eared
u u iian n ly on a slide. A bo ut 5 —6 different areas o f
the sk i:i should he sam pled, i n d u d in g the skin over
the buttocks, forehead, chin, cheek and ears. T h e
smears are stained by the Z ie h T N e e ls e n technique
u rin g ? % in ste a d o f 7 0 % s u lp h u r ic a r id for
dccolourisation, Biopsy o f the nodular lesions and
thickened nerves, and lym ph node puncture may
be necessary in some cases.
T h e smears are graded, based on the num ber o f
bacilli as follows:
1 - 1 0 bacilli in 10 0 fields * 1+
1 - 1 0 bacilli in 1 0 fields - 2+
1 - 1 0 bacilli per field - 3+
1 0 -1 0 0 bacilli per field = 4+
1 0 0 -1 0 0 0 bacilli per field - 5+
M ore than 10 0 0 bacilli, clumps = 6+
and glob i in every field
 * Mycobacterium III: M. Leprae »
T h e b acterio lo gical (bacterial) index ( B I) is
calculated by to ta lin g the num ber o f pluses (+s!
scored in all (he smears and divided by the number
o f smears, T h u s , if eight smears examined have a
Total (if -.ixteen p lu se s, the E l w ill be 2 . Fo r
c a lc u la to r B I, a m ini muni o f four skin k > n iis , a
nasal swab and both the ear lobes have to he
examined.
T h e m orpliological index (M I) is expressed as
the percentage o f uniform ly stained bacilli out o f
the total number o f bacilli counted.
M ouse foot pad inoculation has hern reported
to be m ote sens!rive than skin slit sm ears for
detection o f lepra b acilli in tissues. B u r this is
unsuitable for routine diagnosis and feasible only
for drug poteoev or resistance testing and research
studies.
D e t e c t io n o f a n t ib o d y a g a in st A f. Jeprae
phcnoiic glycollpid antii^en has beer ch irre d to be
a specific d iag n o stic test. A tte m p ts to develop
m o lecu lar d iag n o stic m ethods arc in progress.
M eanw hile, microscopic demonstration o f lepra
b a c illi and h isto lo g y rem ain the mosc useful
dingpnFfi.' procedures.
T r e a t m e n t : D a p s o n c w as the first effective
chemotherapeutic agent against leprosy. Its use as
a m o n o th e ra p y for several years led ro the
development o f nr-istant strains n f lepra bacilli. In
view o f this, multiple drug therapy ( M D T ) is now
recom m ended in leprosy* as in tuberculosis. T h e
c u rre n r r e c o m m e n d a tio n s for p a tie n ts w ith
paucibacillary lesions (T, J 1, E T ) is the Concurrent
adm inistration o f rifam picin 600 mg once a month
and d n p so n d (K ) m g d:Lily for fix mo orbs. Fo r
m u lti b a c illa r y le sio n s (B B , BL, l . l . i, th e
reco m m e nd atio n is rifa m p icin 6 0 0 m g once a
m onth, d.ipsoiiu 10 0 mg dudy and clofazimine 50
mg d aily for two years nr until s i;in smears are
negative. Ethio nam ide or prothionam idc may be
37 S
added to this ne^imen or substituted lor clofazimine.
A m in im u m follow * up o f fou r years fo r
pauc ibacillary and eight years for m u ltib a cillaiy
cases w ould be necessary to detect any relapse.
A n immunotherapeutii: vaccine (M ycobacterium
W ) d e v e lo p e d at th e N a t io n a l In s t it u t e o f
Im m unology, New D elhi is churned to enhance the
effect u f M D T
P r o p h y la x is : C a se finding and adequate therapy
have been the methods employed for prophylaxis.
L o n g -t e r m c h e m o p ro p h y la x is has g iv e n
encouraging results in child contacts o f infectious
cases in In d ia and the Philippines.
There is some degree o f am ij-enic relationship
between the lepra and tubercle bacilli. It is an o ld
clinical ohservacinn that leprosy and tuherculn--.:s
do not usually coexist. B C G vaccine was observed
to induce lepromin positivity and hence its use in
the p re v e n rio n o f le p ro sy w as su g g e ste d by
Kcntandcz as early as in 19 3 9 , Shepard found that
lepra baciili did not m ultiply in the footpads o f
m ice im m unised with B C G . Controlled trials gave
divergent results, from high to no protection. Fie ld
trials with different le p ro ^ vaccines ( B C G + killed
lep ra b a c illi; 1 C R C b a c illu s ) have not p iv e n
conclusive results so far.
MYCOBACTERIUM LEPRAE MURIUM
T h is is the causa live agent o f rat leprosy. It was
first described by Stefansky in 19 0 1 at O dessa. It
has b e e r su b se q u e n tly reported from several
c o u n tr ie s . R a t le p ro sy is c h a ra c te ris e d by
su b cu ta n e o u s in d u ra tio n s , ly m p h adenopathy,
em aciation, ulceiadona and loss o f hair. A c id fast
b acilli resem bling lepra bacilli are found in the
lesions in large num bers. H ow ever, the disease
differs from hum an leprosy In rhat the nerves are
not affected. M . leprae and Af. feprae m urium are
not closely related by D N A studies.
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 Spirochetes
Elongated, m otile, floti hlc bacteria twisted spi rally
along the long axis ire termed ‘spirochetes' (from
Spam* meaning coil and L/j.ure. meaning hair), They
are structurally more complex than other bacteria.
A charsLcrerisric feature is the presence o f varying
numbers oh enda/Jagielfa^ w hich are polar flagella
wound along the helical protoplasmic cy lin d e r and
situated between tlie outer membrane and cell wall.
Endothgcfte arc believed to be responsible for
m otility hut the exact m echanism s o f locom otion
are not understood.
Spirochetes vary widely in size, some being as
long as 500 pm and others as short as 5 p m -T h e y
:irc f l r a n n e g a tiv e , M a n y arc f r e e -liv in g
saprophytes, while some u e obligate parasites.
T h e y m av he aerobic, anaerobic or facultative.
Reproduction is by transverse fission.
Spirochetes belong to the order Spirochetales,
com prising two fam ilies— Sp irn eb et accne
co n tain in g the genera Spirochacta, Cristmpirs,
Treponem a an d Borrelia ; and L e p to sp ira ce ae
containing the genus Leptospira. H u m an pathogens
are found in the genera Treponema, Bitrrclt.i ami
L-cptospira, M em bers o f the genus Spnx-fw cfu a re
SupittphytcS found in water and Srw iigt, w hile
Cristispira are found in molluscs.
Treponemes frrepos, m eaning to turn, and neina,
m e a n in g th read ) are re la tiv e ly sh o rt hlen d er
spircK' hetes wi th ti ne spi r.tls and poi nted or mu ruled
ends. Some o f them arc pathogenic, w hile othera
occur as commensals in the mouth, intestines, and
genitalia. Pathogenic treponemes have n<n been
successfully cultivated in cell free m edia though
the commensals m ay be grown in artificial inertia.
Treponemes cause the following diseases in
hum ans;
L Venereal syphilis caused by T. pallidum
2. E n d e m ic s y p h ilis ca u se d by T. pallidum
[T. endeaticum )
5. Yaws caused by r perferiue
4. Pinta caused by T e in re u tn
T h e y arc almost identical in their morphology,
antigenic structure and ocher features, though there
are differences in the clinical features and natural
history' of the discuses they produce. It has been
suggested that the pathogenic treponemes represent
only evolutionary variation! o f a single species and
that the diseases caused by them, though different
c lin ic a lly an d e p id e m io lo g ic a lly , s h o u ld he
co n sid e red parts o f a co n tin u o u s sp ectru m o f
afo s r j. A c c o rd in fly , the species 71
p a llid u m is now co n sid e re d to in clu d e three
subspecies— subspecies pallidum causing venereal
syphilis, endejnim rn causing endemic syphilis and
perterruf causing yaws.
TrcyfcuwmffpidhdhnT, the causative agent o f syphilis,
was discovered by Schaud inn and Eiotfmann ( 1905)
in the ch an cres and in g u in al ly m p h nodes o f
syphilitic patients, T h e name paliidu jtj refers to its
pale hi a ini ng.
It is a thin, delicate Spirochete
with tapering ends, about 10 pm long (range 4 - 1 4
pm) and 0 .1 -0 . 2 |jm wide. It has about ten regular
spirals, w hich arc sharp and angular, at regular
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intervals t shout 1 pin. It ih .k'Mi'tlv motile, There have been manv -. : ■ o f cultivation of
exhibiting rotation around the Iongax]sf backward T. p a llid u m in c u ltu re s b ir n o n e has been
and forward movement^ and flexion oJ the vdkmtc s u b s ta n tia te d . The iso la te s have h e rn
body. During morion, secondary curves appear and nonpathogcnic treponcmcs, . morphological
disappear in succession bur the primary' spirals are and antigenic sim ilarities w ith 7, pallidum . T h e
unchanged (Kig. 42 . 1 ), bc?t known o f these is the ■'■■■ r, ■strain, w hich has
T. p a l l i d u m cannot he Seen under the light been w idely used as the - i n group specific
microscope in wet films hut can be made our In' treponemal tests tor I he 1 of syphilis. Th e
negative staining with Indian ink. Its morphology K cite i trepnnem e grows w ell in th io g ly co lla tc
and motility van he seen under the dark ground or m edium containing s e r u m /I’hc Reiter treponeme
phase contrast microscope, it doe- not take ordinary is now c la rifie d as T. \ denis
bacterial stains but stains light rose red with . . T pallidum is ■ delicate, being
prolonged d e rm a staining. It can be stained bv readilv inactivated by drying of i v heat ( 4 1 -4 2 * C
silver impregnation methods- Fnnfan/s method is inone hour}. Hencefom ires arc f fifth importance
useful for staining films and Levadiri's method for in the transmission o f infection. $ i-. ■ 1 1 1 ■ lir-. o f T
tissue sections. riaiAJujrj tO heat was thebasi'- ‘fever therapy1
Ultmtruchirally, the cytoplasm of T, pallidum for syphilis. Tr is killed in \~3 ■■■■• ■ at 0 - 4 K l, so
is surrounded by a trilam inar cytoplasm ic that transfusion syphilis can be pi venter by storing
membrane, enclosed by a, cell wall containing blood for at Ieast four days i n i ' before
peptidoglyean which gives the cell rigidity' and transfusion. Stored frozen a t - 7 0 ( in 1 0 % glycerol,
shape. External to this is- the lipid rich outer or in Ingrid nitrogen ( - 1 3 0 ( it remains viahle
membrane liver. Originating from each end of the for 10—15 years. It (■ inactivated by contact with
cell, three or four cndoflagella wind round the oxygen, distilled water, soap, . mercurials,
axis of the cell in the space between the cell wall bism uth, comm on ->n:. -: ■ ■: and antibiotics.
and outer membrane layer; to interdigitate at its - The ant genu structure
centre. Unlike the flagella of other bacteria, these
endullagella do not protrude outsider bur remain
within the outer membrane layer.
Saprophytic spirochetes are generally coarser
m ap^M-amncr, lack the uniform spirals with regular
spacing, and show [ashing motility,
<" i Pathogenic ire pone mes do not
grow in artificial culture media. Lim ited growth
of T- pallidum has been reported in cocullivutioD
with tissue culture Cells. It :is possible EO maintain
T. pallidum in m otile .tod virulent form tor 1 0 - 1 2
days in complex media under anaerohic conditions.
Virulent T. pallidum strains have been maintained
For m any decades by serial testicular passage in
rabbits . One such strain (N ic h o ls strain] isolated
from the br.iin of a fatal case of genera! paralysis o f
the insane in 1912 is still being propagated and
used for diagnostic and research purposes.

C o p y rig h te d m aterial
Hidden page
prim ary lesion heals. D u rin g ibis interval the patient
is asymptomatic. T h e secondary lesions axe due to
widespread m ultiplication u l the Spirochetes and
their dissem ination through rhe blood. Rnrenllar
rjt papular skin rashes„ m ucous patches in the
oropharynx and condylomaca at the mucocutaneous
junctions arc rhe characteristic Ic'-iions. Spirochetes
arc abundant in the lesions and consequently the
patient is most infectious d u rin g the secondary
stage, 'Hiere may also be ophthalm ic, osseous and
m eningeal in vo lvem en t. Secondary lesions arc
h ig h ly v a ria b le in d is trib u tio n , in te n sity and
duration but they usually undergo spontaneous
healing. in so n if instances taking as long as four or
five years.
After the secondary lesions disappear, there is a
period o f quiescence known as 'latenr syphilis".
D iag n o sis during this period is possible only by
serological tests. Jn many cases, this is followed by
natural cure but in others, after several hears,
m anifestations o f tertiary syphilis appear. Th e se
c o n s is t ut c a rd io v a s c u la r le s io n s in c lu d in g
aneurysms, chrom e grunuloniata (g u m m au ) and
m eningovascular m anifestations. Tertiary lesions
c o n ta in few s p iro c h e te s and m ay rep re sen t
manifestations (it delayed hypersensitivity. In :l few
cases, neurological m anifestations such as tabes
dorsalis or general paralysis of the insane develop
several decades after the initial infection. These arc
known as late tertiary or quaternary syphilis.
In s y p h ilis a c q u ire d n o n v e n c re a lly (as
occupation ally in doctors or nurses), the natural
evolution is as in venereal svphilis. except that the
p rim a ry chancre is evtragenital, usually on the
fingers. In the rare instances where syp h ilis is
transm itted by b loo d tran sfu sio n , the p rim ary
chancre Jo e s not occur. In congenital syp hilis,
where infection is transmitted from mother to ferns
transplaccntally, the manifestations and course JUT
different. Trans placental transmission can. take place
at any Stage uJ pregnancy. A w om an iv ilh early
syphilis can infect her fetus much more com m only
( 7 5 -9 5 per cent) than one with syphilis o f osier
two years duration {.15 per cetit). The lesions of
congenital syphilis usually develop o n ly after the
fourth month o f gestation, the time when fetal
Immune competence starts apj.taring. This suggests
that the pathogenesis requires immune response
from the fetus, Congenital syphilis can be prevented!
if the mother is given adequate treatment before
th eto urlh month ni pregnancy.Hie ohs-tetric history
in an untreated syphilitic woman is typically one
,1 bullions and stillbirths followed by live births
o f iniants with stigmata o f syphilis and finally of
healthy infants.
As the manifestations
o f syphilis are p r o te in and as there are
asymptomatic periods during the natural course of
the disease, laboratory aid is essential for the
diagnosis of the disease. It is also important in
assessing the rune alter treatment, Because o f the
social and emotional overtones of the disease, the
diagnosis of Syphilis should impose a great sense
of responsibility on the laboratory. Laboratory
diagnosis co n sists o f demon strati on of ihe
spirochete* under the microscope and of annbodies
in serum or CSF.
D iagn osis by m icro sco p y is
applicable in primary and secondary stages uthI in
cases o f congenital syphilis with superficial lesions.
Specimens should be collected with care as the
lesions arc highly infectious. The lesion is cleaned
with a gauze soaked in warm saline and the margins
gentlv scraped so that the superficial epithelium is
abraded. Gentle pressure is applied to the base of
the lesion and the serum that exudes is collected
preventing admixture with blood, Wei films are
prepared with the exudate and after applying thin
c overall ps, exam ined under the dark ground
microscope. 77 paflkhnzi is identified by its slender
spiral structure and slow movement. Differentiation
from saprophitic spirochetes commonly present in
the genital area needs experience.
Dark ground examination is useful hut negative
results do not exclude the diagnosis o f syphilis,
because o f Its low sensitivity. A treponem al
C o p y rig h te d m ateri
 4 SpirTCUfttea ►
ro nctTitrutign o f \Q* per m l in the exudates is
required for the test to he positive.
T h e direct fluorescent antibody test for J . pallidum
( U F A - T P ) is a better an d safer m e th o d for
m icroscopic diagnosis. Smears o f the exudate arc
fixed with acetone and sent to the Laboratory, where
the D F A - T P test is done using fluorescent tagged
ant] - 7i p&llU h; rr.1 antiserum . T h e use u f specifit:
m o n o clo n a l a n tib o d y has m ade the test m ore
reliable-
S e r o lo g ic a l te sts; T h e se tests fo rm the
ma-.ustay o f laboratory diagnosis, A large number
o f Tests have- been described, o f w hich only a few
am now employed -
Serological tests for syphilis may he classified
as follows:
1. tests for anribodies reacting w i:h card io lip in
antigen rcagin tests; standard tests for syphilis;
STS
2. tests for antibodies reacting w ith group specific
trcpnnenutl antigen
3. tests for sp e cific a n t: bo dies to p ath o g en ic
treponema ( T pallidum).
1, jR c jg in anybody tests These tests use the lipnidaL
or cardiolipin ami gens and arc known as 'standard
tests for syphilis’ or S T S . (T h e antibody reacting
w ith cardiolipin is known an rcagin. T in s can he
m isleading, as the 7 g F antibody in atopy is also
calle d rcag in , though there is no co n n ectio n
between the two.)
T h e first o f the reagio antibody tests was the
W asse rm an n com plem ent fixation test U 9 0 G ),
w hich oci^i natly used a w atciy extract o f the liver
o f a syphilitic ferns as the antigen. Ttn-i was later
substituted by an alcoholic extract o f ox heart tissue,
to w hich Lecithin and cholesterol were added. T h is
crude extract was su b seq u e n tly replaced by a
p u r ifie d lip id e x tra ct o f b e e f H eart (c a lle d
cardiolipin), w ith added lecithin and cholesterol,
as s ta n d a rd is e d by E a n g b o rn ( 1 9 4 5 ) . T h e
com plem ent fixation test remained the principal
serological test for syphilis, till it was replaced by
the sim pler flocculation tests w hich also use the
381
cardiolipin antigen, T h e W asserm ann test is no
longer in use now.
T h e first flocculation test used w \Lcly was the
tube flocculation rest o f K ah n . T h e K a h n test has
been replaced by the simpler and more rapid V D R L
test, w hich gives mote quart i rat Lvt results ( V D R L ,
for Veneral Disease Research Laboratory, U S P H S ,
N ew V ir k , where the test was developed). In the
V D R L test, the inactivated serum (that is, scrum
heated at 56 UC for 30 m in u t a ) is m ixed v. rh
cardiolipin antigen on a special slide and rotated
for four minutes. C a rd io lip in remains as uniform
crystals in normal serum hut forms visihle d u m p s
on com bining w Ah rcagin antibody T h e reaction
is read under a low power microscope. B y tesring
serial dilutions, foe antibody titne can be determined.
T h e results arc reported qualitatively as 'reactive',
'weak reactive1 or 'nor rcacrivc’. Fo r quantitative
re|Jortmgj the reciprocal o f the end point is given
as tire litre, for example 'reactive 4 d ilu tio n 1 or
titre 4’.
V D R L test can be used for tesdng C S F also,
but not plasma. C S -F need not be heated prior to
foe test.
A number o f m oc.fications o f the V D R L test
have been developed, o f w hich the Rapid Plasm a
Rcagin ( R P R ) test is the most popular. T h e R P R
rest uses V D R L antigen containing fine carbon
particles, w hich make the result more clear cut and
evident to the naked eye. R P R test can be done
w ith unheaccd scrum or plasma, but is not suitable
for testing C S F . Autom ated R P R test ( A R T ) is
available for large scale tests. A n automated V D R L -
E L 1 S A rest has been devehiped w hich can measure
I g C and lg M antibodies separately and is suitable
for large scale resting o f sera.
As candioii])in andgen is present both in T p aU id b m
and in mammalian tissues, re agin an I ibtHUes may
be induced by treponemal or host ti-isue antigens.
T h is accounts for foe biological false positive (B F P )
reactions, which constitute foe major disadvantage
o f S T S . IJ F P reaction* are defined as p o s itiv e
reactions obtained in cardiolipin tests, w'.th ncgai"ive
C o p y rig h te d m aterial
results in specific treponemal tests, in the absence
ot past or present treponemal infections— and not
cau se d by t e c h n ic a l fa u lts , T h e y represent
nontrejioneniu] c.Lrdiuli|nii antibody responses,
B F P reactions may occur :in about one per ccnr
o f normal sera. B F P antibody is usually Ig M , w hile
reajd [' ai ilibody 1 1l syphilis is m ainly Ig G . C Ji ilies I ly,
HI'"!1 reactions may be classified as acute nr chronic.
A cute B I T reactions last only for a few weeks or
months- and arc nsnaH y associated w ith uiiute
infections, injuries o r inflam m atory conditio ns.
C h ro n ic B F P reactions persist for longer than six
months and arc typically seen in ST,F, and otHer
collagen diseases, Leprosy, malaria, relapsing fever,
infections iMononucleosis, liepELlhis and tropical
c n s in o p h ilia are rx iim p lts o f other co n d itio n s
associated w ith H I11" reactions.
Rcagin antibody becomes detectable 7 - 1 0 days
after the appearance o l prim ary chancre (nr H—5
weeks ntfL-r acquiring the infection).T h e sensitivity
in the prim ary stage is per cent with the
Citrus being low, upco eight. In the secondary Stage,
the sensitivity is 10 0 percent and tit res mngc from
1 6 to 12 8 or more. Prozonc phenomenon may be a
problem in high titre sera and it is therefore
essential to te^r sera in dilutions.. Another stage of"
s y p h ilis in w h ic h such h ig h litre s arc seen is
congenital syphilis. After the secondary stage, tit res
d im in ish and about a third o f patients, w ith late
syp h ilis are seronegative* T h e firtCS may rise in
patients developing cardiovascular neurological or
gummatous lesions. In some cases ofneunwrypliiliH,
rcagi n, tests m ay bo negative wi rh scrum but pi isi ti\'c-
with the L ’ SF, Reagm teste usually become negative
6 - 1 8 months after effective treatment ot syphilis,
depending on the stage at w hich treatment is given.
I towever, if treatment is started lare, the tests may
rem ain positive in low litres.
In order to avoid
B K P reactions, rc^ts using cultivable treponemes an
antigens were developed: These employed e] ll- Reiter
trepo nemes (originally believed to be an adapted
strain o f 7. pallidum ). T h e test most com m only
em ployed in this group was the k e im r protein
c o m p le m e n t fix a tio n ( R P C F ) test, u s in g a
lipopolysaccharkic-protciii complex antigen derived
from the treponeme, Its sensitivity and specificity
were lower than, those o f tests using T. j u J litfam.
T h o u g h R P C F w as g e n e ra lly free fro m B F P
reactions* it still gave some false positive reactions.
R P C F and either Reiter tre] Minetrie tesCKare not DOW
in general use.
T h e se tests Line the
virulent N ic h o ls strain o f T pallidum maintained
bv serial inoculation in rabbit testes.
T h e first in th is g ro u p is the T re p o u e m J
pallidum im m obilisation ( T P I] test intRxluccd in
194 9. T h e tost serum is incubated w ith complement
and T p.illiilum m aintained in a. complex m edium
a n a e ro b ica lly . I f a n tib o d ie s arc p re se n t, the
trcpotiem es are im m o b ilise d , that is, rendered
ro n in o tile , w hen exam ined un d e r d ark ground
illum i nation.
In its rim e, T P I w as the most specific rest
available inr diagnosis ttl syphilit ami w in considered
the gold standard in syphilis serology. How ever,
because o f its extreme complexity, it was available
only in a tew laboratories. T h e T P T test has now
been supplanted hy nrher tests such as F T A - A B S
and T P I ]A w hich are quite as specific and m uch
simpler.
T h e fluorescent treponemal antibody (E 'T A l test
is an indirect im m unofluorescence test using as
an eigen, smear? prepared on slides w irh N ic h o ls
Strain ol 7. polfuium . T h e slides can be started for
several m onths in deep freeze. T h e currently used
m odification o f the test is the F T A -a b s o r p tio n
( F T A - A B S ) test in w h ic h the test scru m is
p re a b so rb e d w it h a so n ic a te o f the R e ite r
CtfeponeTnes (sorbent) to elim inate group specific
reactions, F T A - A R f i is [in specific as the T P I test
and is now accepted as a standard reference test.
Ilo w tV e r,.ix it can be done only in suitably equipped
Laboratories-, it is nor available for ton fine resting.
T h e T. p:\lliihtm h e m a g g lu tin a tio n A s s a y
CTP] IA ) uses tanned erythrocytes sensitised with
C o p y rig h te d m aterial
a sonicated extract at T. pallidum, as antigen. T h e
p ro c e d u re now e m p lo y e d is a m ic r o -
h e m a g g lu tin a tio n test ( M . H A - T P ) , w h ich is
capable id'being automated.
T h e test sera for T P H A are absorbed w ith a
d ilu e n t c o n ta in in g co m p o n e n ts id the R eiter
frcponcmc, rabbi t testis and sheep ervth rocytes- Sera
are screened in an initial dilution o f 1:8 0 but ritrcs
o f 5 1 2 0 or more art co m m u n ln the secondary stage.
T P H A is just as specific as F T A -A B S and almost
as sensitive, except in the prim ary stage. Jt is also
m uch sim pler and more econom ical. N o special
equipm ent is needed. him are available
commercially. Th e se advantages have made T P H A
a standard confirm atory test-
T a b lc 4 2 .1 shows the relative sensitivities o f
the serological rests in comm on use.
lin ^ v m e im m u n o assays. ( E l A ) h ave been
developed u sin g T. pallidu m antigens and are
available conn merci ally ( B io -L n z a Bead lest; Captia
S v p h ilis -G test). A rjjrid agglutination test has been
developed, using latex particles coated with three
im m unodom inant proteins of T p a llid u m , obtained
by recombinant technology'. It is claim ed to be as
specific as T P J 1A* and more sensitive.
T h e p ra ctice for se ro lo g ica l scre e n in g lo r
syphilis varies in different countries. In the U K , a
com bination ot V D R I . and J P H A Letts l:- Used.
T h is is an efficient com bination for the detection
or exclusion o f syphilis at all stages, except the early-
prim ary stage. A repeat teat 1 - 3 m onths later w ill
bring even this no light, In the U S A , screening is
by V D K L or R P R test alone. T h is may fail ro detect
about one per cent o f secondary syphilis due to the
proaone effect and about 30 per cent o f latent or
Laic syphilis.
Primary 70--SO 8 5 -1 0 0 65-85
Secondary 100 100 100
g
o
l.atcnt/late 9 5 -1 0 0 95-100
■/o
i
Q uantitative tests are useful in m onitoring the
patients response to treatment, indicating the Stage
o f the disease and in detecting reinfection. Reagin
rests are preferred because they usually becom e
negative following treatment. Lf treatment is given
very early, the serum may not liecome positive at
a ll- T re a tm e n t in th e p r im a r y stage leads to
scnoreversal in about four months; in the secondary
and early latent ■ stages, it takes 1 2 - IS m onths; in
later stages, it may take five year* or more. In some
cases low titre reactivity m ay persist indefinitely, in
spite o f effective treatment. Specific treponemal tests
are o f little value as indicators o f clinical cure, as
they tend to remain positive in spite o f treatmenr.
T R H A titres may fall rapidly M i t r i n g treatment
in secondary syphilis but remain positive for life in
low titres.
T P H A and F T A - A E 5 are helpful in excluding
o r co n firm in g the diagnosis o f syphilis and fi?r
identifying H t ? reactions. T h o u g h false positive
reactions were believed u> have been elim inated
with the introduce ion o f these specific rests, it is
not truly so, Both T P H A and F T A - A B S can give
false po sitive re su lts, th o u g h very rarely'. AIL
serological tests for syphilis may be positive in
non venereal irepone mi .HOses, and some in 1 few
other spirochcatal infections as w ell. In L y m e
disease, V D R L test is negative,but F T A - A B S m ay
be positive.
A n eg ative r P H A v ir t u a lly e x c lu d e s the
diagnosis o f syphilis, past or present, except in the
very' earlv stages. In ncnrosyphilis, a negative C S T
V D R L test may not be conclusive but a negative
T P H A test e lim in a t e s [he p o s s ib ilit y o f
iieurosyphilis.
D etectio n o f specific Jg M an tib o d y m ay he
helpful in some situations. Being the initial] type o f
antibody to appear, Ig M is detectable by the second
week o f infection. IgM antibody production ceases
soon after elim ination o f infection by treatment.
Persistence o f Ig M antibody indicates continuing
active disease and the need for treatment. A s Ig M
does not cross the placenta, its presence in neonatal
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scrum co n firm s co n g e n ita l sy p h ilis an d h elps
differentiale it from senipositivrtv due to passively
transferred m aternal antibody (iyphiloto xem ia).
M in v techniques have been developed tor the
selective detection o f Ig M antibodies, These include
modifications o f rhe F T A - A B S , T P I I A , E 1 A and
V D R L rests, u sing whole sera or separated Ig M
fractions. W h e n such te^-ts are nor available, parallel
tests o f maternal and neonatal s e n may settle the
d iag n o sis o f co n g e n ita l s y p h ilis, in w h ich the
neonatal serum m ay show a higher titre o f anti Innh
than rhe m.Lfcmal scrum - Serial testing is alsotiadail
because the litre o f passively transferred antibody
decreases ra p id ly , rhe V D R L test b e co m in g
negative by three m onths.
Venereal syphilis is w orldw ide
in distribution. D u rin g the five centuries that irh as
been re c o rd e d an d s tu d ie d , the d ise a se has
undergone much variation Lii its natural history and
clinical feirures. A s originally described, ir was a
h ig h ly v iru le n t disease w irh flo rid cutaneo us
manifestations. W irh the discovery o f the dramatic
therapeutic response to penicillin, if was hoped that
it may even be possible ro eradicate syphilis, as the
disease has no extra human reservoir. However, not
on Iy has it nor bee n pnss ible to c I i m i rate the d i seasc
but an increase has occurred in iis incidence, due
ro the ch an g in g custom s, habits and values in
society.
T h e advent o f the A I D S pandem ic has had ait
im pact on syphilis. In most places,, fear o f A I D S
and safer sex practices led to a fall in the incidence
o f syphilis and all S T D h initially, but this trend did
not continue everywhere, Concurrent infection w ith
syphilis and Hi V is com m on and 1nay lend to earlier
evolution o f neUrosyphilis.
T h e im m une mechanisms in syphilis
arc not adequately understood. H um oral immune
response against the tneponcnne docs not appear to
be effective as rhe disease progresses unhindered
for decades even in the presence o f a vigorous
antibody response. C e ll mediated im m unity may be
more relevant. T lymphocyte* and macrophages arc
p re d o m in a n t in e a rly s y p h ilit ic le s io n s .
Specifically sensitised T h l cells secrete cytokines
favouring clearance o f Spirochetes by activated
macrophages.
Reinfections do not appear to occur in a person
already having active infection. Ti was believed that
pirnruJlFtrori or infection im m unity, as seen in some
parasitic infections, holds good in syphilis also and
that a patient becomes susceptible to reinfection
only when h it original intevtinn is cured- However,
it has been shown that some degree o f im m unity
to rcintcction occurs in experimental animals and
persons: w hose in fe ctio n has been co m p le te ly
elim inated by treatment.
A s t r a n s m is s io n is by d ir e c t
contact, it is possible to protect against syphilis by
avo idance o f sexual co n tact w ith an in fected
Individual. W h e n this is not com plied w ith , the
use o f p h y s ic a l b a rrie rs (su ch as c o n d o m s ],
antiseptics (potassium permanganate 1 or antibiotics
may m inim ise the risk. T h e Use o f prophylactic
penicillin carries the danger that it may suppress
the prim ary lesion without elim inating the infection
so that recognition ami treatment o f the disease
may become more difficult- N o vaccine is avadable,
P e n icillin is u n ifo rm ly effective in
syphilis but it is necessary to give an adequate dose
and m aintain the drug level for a sufficiently long
period to establish cure. A -single injection o f 2-4
itiiUion units L>f benzathine penicillin G 1? adequate
in early cases, hbr Late syphilis, this am ount may be
repeated weekly for three weeks. In patients allergic
to penicillin, daxycycliae mav be used. Ceftriaxone
is effective, particularly in ncurnsyphllis Penicillin
treatment in syphilis sometimes induces a reaction,
rhe J a r is c li-lk r x h c im e r reaction, co n sistin g o f
fever, m liaise and exacerbation of symptoms. It is
frequent, but harmless, in prim ary and secondary
syphilis, and can be managed with bed rest atld
a s p irin . It ls rare in late: s y p h ilis b u t can be
d a n g e ro u s in so m e cases o f g u m m a to u s,
cardiovascular or ncurosyphihs. It is believed to be
due to the liberation o f toxic products like tumor
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Hidden page
c h a ra c te ris e d by liy p t r p ig m e n c a tio n
hypopigm C ita tio n . T is s u e s other than skin are
seldom affected.
J|"he causative agent lh T. t-irateum. It is very
closely related to T. pallidum hut is not anti get ically
id e n tica l to cross im m u n ity between pinra And
syphilis is only partial.
Severn! commensal trc]>i>ncmes: otoif on the IulccllL
and genital mucosa and may cause confusion in the
dia^nosihot syphilis hy dark field examln an*hi. IIit y
are a heterogeneous group and have not been
adequately characterised* He si known among them
is the oral spirochete, T! dejifjVofe, which can he
readily cultivated, Trcponcmcs also occur on the
surface of gastric and colonic epithelium in human
beings and animals.
During early attempts t<> grow the $yphtlis
spirochete in cultures, several tfcponemcK had been
grown am i m istakenly calle d 7 ! pallidum — for
example, the Reiter and K azan strains w hich h iv e
been identified as T. pha^edenis n u i tlie ivirulent
N ic h o ls and N o g u ch i strains w h ich have been
recognised as T. refringens.
In experim ental w ork cm T patH dum in
n b b ils , T. paraluiscuniculi (formerly T. cuniculft^
w hich has a very similar Appearance and causes
natural venereal infectio n in rabbits, may pose
problems.
BotfTtli&C are large, m otile, refraLtilc spirochetes
with irregular, w ide, open coils. T h e y arc usually
5 - 3 0 pm long and 0 .3 -0 .7 urn w ide. T h e y tre
readily stained b y o rd in a iy methods and are G ram
n eg ative. Several species o f U o rre lia o ccu r as
com m ensals on the buccal and genital mucosa.
relapsing fever, B. nTrccmr which sometime* causes
fusospirochcrosis and U, buj^do ritri, the causative
agent o f T.ymc disease.
Of
Relapsing fever { R F ) has heen known since rhe
time o f Hippocrates and has occurred in epide m ic,
endem ic or sporadic form throughout the world-
R F is an arthropod-hom e infection, two types o f
w h ich o ccur - louseb<O L t and tick b o m e . T h e
b m rcli.ie iOLUsirg them are in d istin g u ish ab le ii>
morphology and many other teamnes bait differ in
their arthropod hosts.
rhe causative agent <>t epidem ic or lousebome
R F is B. rccim entls, first observed by O berm eier
(1 K7.1) in tlie blood of patients during an epidem ic
in Berlin, It is an exclusive hum ail pathogen, being
transm itted from perapn to person through hody
lice ( A d i W u j Jlujrlajttu m /poris). N o extrabuman
t o c m i r is known,
B arrel iac causing endem ic o r tick b o rn c RF
norm ally live in their natural hosts— rodents or
other m am m als on w hich the vector ticks Iced.
13um an infectio n is o n ly an accidental event.
HorrellLac have been assigned to various species
based on the licks that c a n y them. C h e r ten species
ot boricliae are known to infect human beings and
cause R F (B . diifttJJHJ, B. hetm an, B. p\irkcrir etc.)
T h e y are generally confined to certain geographic
areas. T h e re is evidence from D N A h o m o lo g y
studies to indicate that all o f them may belong to a
■ single species-, with separate host adaptation. T h e
descriptions that follow apply to all o f them, unless
stated otherwise,
B, ica u ren th is an irregu
with one or both ends pointed. It is 8 -2 0 pm long
and 0 ,2 -0 ,4 pm wide. It possesses 5 - 1 0 loose spiral
colls at intervals o f about 2 iu m .lt grains well w ith
G ic m s ia n d bacterial drains ^nd is G ra m negative.
U o rre lia are
iiiidOaerophiJiL:- O p tim um temperature for growth
is 2 8 -3 0 '■'C.. C u ltivatio n is difficult but has been
successful in com plex m edia co n ta in in g serous
flu id s. G r o w t h o ccurs m i the c h o rio a lla n to ic
membrane o f ch ick embryos. For prim ary isolation,
the best m e th o d i.s to in o cu la te m ice or rats
C o p y rig h te d m ateri
LutrapeTLEoneally. W h e n using experimental animals,
great cane has to be eaten to ensure [hut the anitn J s
are free from pre-existing borreliosis.
B o m c lia r e a d ily
undergoes antigenic variations in vivo and this is
believed [O be the reason fur the occurrence o f
relapses in the disease. A ntigenic variations have
been shown to be caused by D f t A rearrangements
in linear plasm ids present in burred a. U ltim ate
recovery after a numher y l relapses may be due to
the developm ent o f im m unity to all the antigenic
variants. Agglutinating, complement fixing and Lytic
a n tib o d ie s develop d u rin g in fe ctio n but their
demonstration IS not jJOvsible as a routine diagnostic
rest due it) the difficulty in preparing satisfactory'
antigens,
A fte r an. incubatio n p erio d o f
2~ 10 days relapsing fever sets in as fever o f sudden
onset. D u rin g this period, borreEiae sire abundant
in the patient's blood. T h e fever subsides in 3 ~ 5 in some outbreaks, a high rate of fatality, tn lice,
days. After an afebrile period o f 4 - 1 0 days during the horrdia js confined to the hemotymph iSnd LS
w hich borreliac arc not dem onstrable in blood, not shed in saliva or excreta, So the infection is
another bout o f fever sets in. T h e horretiae reappear transm itted not by the bite o f Lee but by their being
in blond during the relapses o f fever. T h e disease crushed and rubbed into abraded skin, fi. reorJTenris
ultim ately subsides after d - 1 0 relapses. is not transmitted tran&ovarially m lice.
Experim entally, rodents such as rats, mice and. T ic k b o m e relapsing fever occurs as sporadic
Loss rea d ily, g u in e a p igs m ay be in fe cte d by cases in endem ic areas. It is a "place disease' and is
Lnirape rilonea] injection- BoneUae m ay survive in frequently associated with certain dwellings or ocher
the brains o f infected anim als after they have locations that are inhabited by Infected tic k s ,T h e
disappeared from the blood. disease is m ilder but relapses arc mo-re frequent
L o u s e h o r n c re la p sin g fever than In Lousebome fever. T h e bcmelia persists in
tends to o ccu r as epidem ics w henever poverty, the body o f infected ticks throughout their life and
o v e rc ro w d in g an d tack o f p e rs o n a l h y g ie n e is also transmitted transovarially so that the ticks
encourage louse infestation. E p id e m ic relapsing acr as reservoirs as well as vectors. T h e borrelia
fever used to be very comm on during wars and in i ovaries all parts o f the body o f the tick aod is shed
ja ils of form er days but with im provem ents in in its saliva and fcccs, So the infection is transmitted
hygiene and the discovery o f insecticides, it has [o h u m a n s throug h the bite o f tick s or th e ir
now become rare. It survives in some areas, as in discharges. Several species o f soft ticks belonging
parts ol A frica and appears as outbreaks whenever to the gen vs O m itho do ros act as vectors, different
civ il strike and famine encourage Large scale- louse species being responsible in different regions, in
infestation.The louscbornc disease presents a more In d ia, the vector species are O, thoSoEoni, O, aosai,
severe d ir Leal picture than the tickbom e variety O . lahoeensis and the fowl tick, pcrsicus.
and is associated with jaundice, hemorrhages and, Tliese soft ticks can live for ten years or more with

C o p y rig h te d m aterial
Hidden page
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jlii J
cross Absorption reactions using im m une rabbit
sera or more recently with monoclonal antibodies.
Genetic methods, such .ls restrict ion endonuclease
analysis and 1>NA pairing are used for further
classification into serotypes.
In n a tu ra l re s e rv o ir h o s ts ,
leptospiral infection is asym ptom atic. How ever,
w hen infection is transm itted to other an im als,
clinical disease may result. H um ans arc interred
w hen the lcptospircs in water contaminated by the
urine o f carrier an im ak enters the body through
cuts o r abrasions on the skin or through intact
m u co sa o f m o u th , nose o r c o n ju n c tiv a - T h e
incubation period is usually dtmU JO days (range
2 -2 6 dajra). T h e clinical picture varies from mild
undifferentiated pyrexia to seven? or fatal illness
w ith hepatorenal damage (W e il’s disease). Ln severe
cases, I lie onset is acute, w ith rigor, vo m itin g ,
headache and intense injection o f the eyes. T h e fever
is irregular and usually subsides in about ten days.
Jaundice occurs in about 141-20 per cent oi cases
by the second or third d ay Purpuric hemorrhages
s o m e tim e s o c c u r on th e sk in and m u co sa .
A lbu m in u ria is a constant feature.
T h is t y p ic a l p re s e n ta tio n is u n u s u a l.
Ljepitn&j ■ i n inis is now classi tied into two clinical types
— icteric and nonrcfenc, M any cases present as
a s e p tic m e n in g it is an d in so m e , a b d o m in a l
sy m p to m s p re d o m in a te . C l in ic a l d ia g n o sis is
im possible in the m ajority o f c a w and unlem a
high index o f suspicion is m aintained and laboratory'
assistance sought, leptospirosis w ill be missed in
all hut a few instances.
Lcp to sp ircs are seen in the blood during the
acute phase o f the disease b u t can seld o m be
demonstrated after B~1Q days. T h e y persist in the
internal organs, and. most abundantly in tbe kidneys,
so th:it they may hu demonstrated in the urine in
the Eater stages o f the disease.
Serious cases o f leptospirosis are caused most
often bysem tvpe icternhaemntrhagiae, though they
m ay also be due to Copenhagen! and lens often
bamviae, grippocyphosa, pirogencs mid some others.
A septic m eningitis is com m on in canicola infection
an d a b d o m in a l sy m p to m s in g rip p a ly pho&a
infections. How ever, c lin ica l syndrom es are not
serotype specific and an y type of illness can be
produced by any serotype.
D ia g n o s is m a y he
m ad e b y d e m o n s tra tio n o f the lc p to s p irc s
m icroscopically in blood or urine, by isolating them
in culture or by inoculation of guinea p ig s or by
serological tests.
A s leputtpires disappear
from the b lo o d afte r the firs t w eek, b lo n d
exam ination is helpful only in the early stages of
the d ise a se , b efore a n t ib io t ic s are g iv e n .
Lcptospircs may be demonstrated by examination
o f the blood under the dark field microscope or
by immunofluoreGccftcebut this is o f little practical
value.
T h re e or four drops o f blood are inoculated into
each o f several bijou bottles containing E M j f i
or sim ilar m edium , t he buttles are incubated at
.17 ^ for two days and left thereafter at room
temperature in the dark for two weeks. Samples
from the cultures am examined cvctv third day
for the presence o f lepiospirta under dark ground
illum inatio n. Prim ary isolation may be delayed
and may take many weeks to months. Chances of
isolation are increased by culturing blood daily at
the early stage o f the disease. Leptospires may
sometimes be isolated from tbe C S F also.
T h e blood from the patient is also inoculated
intraperitoneally into young guinea pigs. W it h
virulent serotypes like tcterohaemorrhagiac, the
anim als develop fever and die w ithin S—12 days
w ith jaundice and hemorrhage into the lungs ami
serous cavities. W it h other serotypes such
canicola and pom ona the animal may not become
111 and infectio n w ill have to be id en tified by
demonstration of the lcptospircs in the peritoneal
fluid, by blood culture or by serology. From the
third day after inoculation, die peritoneal fluid is
exam ined daily under dark ground illum ination
and w hen leptospires are detected, the hlood
C o p y rig h te d m aterial
withdrawn by cardiac puncture is inoculated into
culture media.
Leptosplres appear in
the urine in the second week o f the disease and
intermittently thereafter tor 4 -6 weeks. T h e urine
should He examined immediately after voiding an
leprospires readily undergo lysis in acid urine.
Centrifuged deposit o f the urine may he examined
under dark gnuind illum ination. Direct culture of'
u rin e is se ld o m s u c c e ss fu l b ecause o f
contamination but isolation is usually possible by
inoculation into guinea pigs-.
T h e identification of the isolates of leptnspires
is made by agglutination w ith type specific &cra.
D u e tit the large number o f serotypes anti, the high
degree o f antigen ie cross, reactions between them,
ide nti fitari on o f isolate* is a compl i cated procedure
and is generally confirm ed hy one of the W H O /
F A O Reference I aboratorics.
A n tib o d ie s appear in
scrum towards- the end o f the first week o f the
disease and increase till the fourth week, declining
th e re a fte r, A g g lu t in in s m ay, ho w e ve r, be
demonstrable years utter the infection. Tw o types
o f sero log ical tests arc availab le , the broadly
reactive screening tests and the serotype specific
tCStSr
T h e broadly reactive or genus specific tests identify
Leptospira! infection without indicating the exact
infecting scrovar, T h e antigens tor these tes-cs are
prepared from the nonpathogenic L . biflexa Panic
1 strain. T h e tests em ployed include sensitised
erythrocyte lysis ( S F L ) , cu m p lem cat fix atio n ,
agglutination and indirect immunofluorescence-
E L I S A Has been used tn detect Ig M and Ig G
antibodies separately, in order ro indicate [he *rage
o f infection. A simple and rapid d ip -stick assay
has been developed Inr the assay o f Icptospira-
s]:CCl1 ic Ig M antibody ill hum an sera.
T h e type specific tests identify' the infecting
serovar Hy d em o nstrating specific antibodies.
M acroscopic and microscopic agglutination rests
are u sed fo r [Ills p u rp o s e . In tbe fo rm er,
formal] nised suspensions o f prevalent lepto&pina
seruvars are rested for macroscopic agglutination
w ith serial d ilu tio n s o f the test scru m . T h e
microscopic agglutination test ( M A D generally
uses live cu ltu re s o f d iffe re n t serotypes and
agglutination is observed under the low power
dark field microscope. T h is test is more specific
and is usually done only in reference laboratories.
D u e fn the presence o f som e degree o f cross
reaction between different serovais, agg lutinin
absorption tests may sometimes become necessary
for accurate diagnosis.
Infection
in rodents and other animals- may be diagnosed
by sero lo g icnl tests o r by cu ltu rin g pieces o f
kidneys.
I f a shaved and scarified area o f the
skill o f a young guinea pig is immersed in water
for au hour, infection takes place through the
abrasions.
L e p to sp iro sis is considered to
be the most widespread o f r^oonoscs, being regularly
p rese n t in a ll c o n tin e n ts except A n t a r c t ic a .
Pathogenic lcptospires survive for long periods in
the convoluted tubules o f the kidneys in natural
hosts, m ultiply and are shed in the urine. A n im al
Laniers otter excrete upto 1QQ m illion leptospircs
per ml o f urine. If the infected urine contaminates
the water or mud that is neutral or slightly alkaline,
the leptospires survive for weeks. W h e n people
come into contact w ith sairh water* the Icptospires
enter the body through abraded skin or m ucosa
,md initiate infection. C e rtain occupational groups
such as agricultural workers in rice err cane fields,
miners and Sewer cleaners arc more often exposed
to infection, and so leptospirosis is more com m on
in [hem. Leptospiras m ay he shed in the m ilk o f
lse rating animals, However, they die rapidly in m ilk,
and hum an infection through m ilk is not known.
T h e y are not shed in saliva, and so animal bites are
not infectious. Arthropods arc not known to transmit
the infection.
C o p y rig h te d m aterial
 S e v e ra l a n im a ls act as c a rrie rs - R a ts arc
particularly im portant as they are ubiquitous and
carry the m o st p a th o g e n ic sero type i etc r o ­
ll aemorrhagiiie. F ie ld m ice carry gfipporyphosa,
pigs ppmona and dugs Canicula serotypes.. However,
the name serotype m ay He carried hv different
m am m als and one m am m al m ay ta rty different
se ro ty p e s- W h ile le p to s p irc s a rc g e n e ra lly
nonpathogciiic in the reservoir anim al, leptospirosis
is o f veterinary importance as infection o f cattle
and pigs cause considerable economic loss. Infection
am ong a n im a ls is also transm itted by u rin a ry
contam ination o f water and fodder. H u m a n beings
are an aberrant or end' host. There is no evidence
that hum an patients infect others-
Fro m being predom inantly a rural disease of'
agricultural workers* leptospirosis has, in recent
rim es also becom e an u rb an p ro b le m in the
d e v e lo p in g c o u n trie s , T h is is perhaps* doe to
o v e rc ro w d in g , in s a n ic a t io n , in c re a s in g rat
population and the habit o f walking barefooted.
A s le p to s p iro s is re su lts from
contact o f skin or mucosa with contam inated water,
general measures o f prevention such as rodent
control, disinfection o f water and the wearing o f
protective clo th in g contribute to its prevention.
Vaccination has been attempted w ith some success
in dogs, cattle and p ig s. Im m u n it y fo llo w in g
v a c c in a tio n or in fe ctio n is serotype s p e c ific .
Vaccination has also been tried in persons at high
risk such as agricultural workers.
Leptospi res are sensitive to penicillin and
tetracyclines, but the treatment to bq effective should
be started early m die course of the disease, ftn icillin
is given IV , 1 - 2 m il Lion units 6 hourly fur 7 days in
serious cases. A m ild Jarisch-H encheirnar reaction
may occur In some, D o xyryclinc 200 mg orally once
a week Is effective in prophylaxis.

lcterohaernorrhagia-E Weil's disease Fever, jaundiee, hemorrhages Rai Worldwide

Canted* Cankola leiier Influenza like, aseptic- Dog Worldwide
menin^ilis
Grippotvphosa Swamp or marsh Fever, piwtintion, aseptic- Held mice Europe, Ainca,
fever ffifctliflgitis 5.E. Asia, U S A

Pomona Swineherd's Fever I^E America,

disease Europe, Middle
East, Indonesia,
Australia
Hebdomad La Seven day fpwr Vever, lympkiLrlKmiiisthy Reid mice Japan. Europe.
USA
Forrbragg Pretihial lever, Fever, rash r*vei ribin N(ir known Japan, S.E.
Port Bragg fever Aaia* U S A

l^yroge nes Febrile Fever Kg S.E, Asia,

spirochetosis Europe, U S A
Bataviae Indonesian Weil's Fever Kat S.E. Asia,
disease Africa, Europe
Hsrdjo Dairy farm fever Fever Cattle U K , U S A , New
Zealand

C o p y rig h te d m aterial
Hidden page
 Mycoplasma
M y c o p la s m a arc a group o f bacteria that arc
devoid o f cell walls and so are highly pleomorphic*
w ith 110 fixed shape or size, T h e y lack even cell
w all p re cu rso rs lik e m u ram ie a cid or
diam innpi iridic acid. T h e cells arc bounded by a
soft trilam inar unit membrane containing sterols.
Because o f their plasticity* they c a r pass, through
bacterial filters and have often been mi stake n far
viruses. T h e first member o f the group was the
organism causing bovine pleuropneumonia, isolated
by Nocard and Roux (1H 98). A similar organism
was found to cause contagious agalactia in sheep.
W h e n many sim ilar isolates were obtained from
animals* human Lyings* plants and environm ental
sources, they came to he called 'pieum pncum aniii-
like nrjipjjiJs'jiJs'tPPLO). T h is tu isitil& tto ry name
has been replaced by the term Mycoplasma (Mycw,
fro m the f u n g u s -lik e fo rm o f the b ra n c h in g
filam e n ts; pLts/nt* d e no tin g their p la sticity o f
shape).
\ 1 yen plasm as have hern placed in the class
M o l ti cutes (literally m eaning soft skin)., order
A Pmft tide.
1 . .Established padrageft;
M. prWufiiorTftie causing pneumonia
2. Presumed p^Thogenm
M. ftQpiinit iiii-1 i ...-rtttb-Tif'Ujn asaLKiaccil with gcniral infecrions
3. iN’oii-parbc^enrc;
M, orate, Mr bufcjile, Af- o fririu m , M - fkidw n in nrophaiynK
Af, Jeone/tnirt^ Af. gcniaiiuiJi, Af. penetrans, Af. pruiurum,
A f spenndtophiium in genual met
B. Siptophjrtie
Achnieplasma hidfami nn tkira and in mouth.
M ycoplasm acales, w h ich co ntains the follow ing
families and genera:
1. F a m ily Afywp/jiirtiaidCtffltf, in w h ich belong
parasitic mycoplasmas requiring cholesterol or
other sterols as an essential growth factor. T h is
contains w o genera:
a. G enus A facoplasma w hich utilise glucose or
arginine but do ro t split urea, and
b. G e n u s C/jneapJasmit w hich hydrolyse urea.
2- Fa m ily A choIcpIuiWiltACCHC, mostly saprophytic
myco plasm is , w hich do not require sterols as
growth factor.
3. Family Spiroplum atam c, contai ning the Genus
Splraphanin, w hich parasitise arthropi>di and
plants. T h e y are sterol dependent. T h e se are
helical iti shape.
4. F a m ily A n a cro p h x m a tscetc, c o n ta in in g the
genus AnacrofJasma* w hich are strict anaerobes*
found in the rumen o f cattle and sheep.
M yco p ksm as may be saprophytic, parasitic or
pathogenic. M ore than 100 species o f mycoplasma
a rc k n o w n to cause d ise a se in a v a rie ty o f
C o p y rig h te d m aterial
r r, -
\ i
MVCOPI.ASMA
m am m alian, Inject and plant host-. A bo ut ■ iijttct.-n
species, belonging In three m i l i n are found m
hum an beings (Table 4 3,\ )r
M y co p la sm a s art; the sm allest
tree'living m icroorganism s, and one o f the most
pleom orphic {1-ig. J 3 .1 ) , T lte y occur as granules
and filam ent1; of various sizes. T h e granules may
he coccoid, balloon, ring or star forms. T h e
filaments are slender, o f varying length* and show
true branching. M ultip licatio n i^ by binary fission,
but as genom ic re plication ami cell divisio n arc
a lie n asynchronous. budding farm s and chains o f
beads arc produced. A distinctive feature seen in
som e species is a bulbous enlargem ent, w ith a
differentiated tip structure, by means o f which the
o rganism s get attached to suitable host cells
carrying neuram inic acid receptor*. T h e y mav L>t=
responsible for the hemadsorption shown by some
species.
M ycoplusuias do not possess spores, flagella or
fim bria. Som e specie5 (.‘■ shibit a gilding m otility.
M vcopEasmas are G ra m negative but are better
stained hy G lc m s a stain.
M ytop]litmus may he cultivated in fluid or solid
media. T h e y .Lie general!y facultative anaerobes,
growth being hetter aerobically- T h e y grow w ithin
a Icm peraruie range o f 2 2 - 4 1 "G , the parasitic
spccies grow ing optim ally at 3 5 - 3 7 "C and the
saprophytes at low er tem peratures. M e d ia for
cultivating mycoplasma are enriched with 2ii per
ce nt horse nr h u n ian serum and yeast extract.
PcmcLUin j-nd thallium acetate are added as selective
ag en ts, T h e h ig h c o n c e n tra tio n o f s c ru m is
necessary as a source o f cholesterol and other lipids.
C o lo n ie s appear after incubation for 2 - 6 days and
arc about 10 -b (W pm in s iz e .I T c colony is typically
biphasic, w ith a 'fried egg' appearance, co n fu tin g
o f a ce n tral o p aq u e g ra n u la r area o f g ro w th
extending into the depth o f the m edium, surrounded
b y a fiat, translucent peripheral u s e (F ig . 4 1.2 ).
C o lo n ics m aybe seen with a hand lens but arc best
studied after staining by Diene* method, h’or this, a
block o f agar containing the colony is cut and placed
on a slide. It is Covered w ith . l caver slip on which
has been dried an alcoholic solution of mcthvlcnc
blue and azure-
C opyrighfecl m aterial
Hidden page
T h e mycoplasma mav remain in the; threat fotf two
or more months after recovery from The disease.
Eaton (1^44) wis the first lu isolate the
causative agent of the discare in hamsters and cotton
m s. ] le was able to transmit [he infection later to
chick cmbivus by utnrtiotic inoculation. Because it
was filterable, it was considered to be ,lvirus (Eaton
agem ), but was subsequently shown to be a
mycoplasma und named A /. pneum oniae.
Laboratory diagnosis o f mycoplasmal primacy
atypical pneum onia m a y b e established either by
is o la tio n o f the m yco p lasm a o r by sero log ical
methods. For isolation, threat swabs or respiratory
secretions are inoculated into mycoplasma medium
containing glucose and phenol red. G ro w th is slow
on prim ary iso latio n and m ay take 1 - 3 weeks.
G ro w th is in d icate d by acid pro ductio n in the
m edium . Af. p m u n o n ix e produces beta hemolysis
and agglutinates guinea pig erythrocytes. C o lo n ics
on agar adsorb errtbroc vtes. T h e hemadsorption is
enzym atic and occurs optim ally at 37 DC . T h e coll
receptors are destroyed by neuraminidase. ll inhibits
ciliary m otility in hamster trachea organ cultures.
M . p /i clpJTi ojtj.ic is u n re lated to o th e r h u m a n
m ycoplasm a? and m ay be identified by growth
in h ib it io n by specific antisera. A s iso la tio n is
difficult and delayed, P C R W5&y w hich is rapid
and specific is being used where feasible.
Serological diagnosis may be made by specific
tests using mytopbiRrmil antigens or hv nonspecific
methods. Am ong the former, immunofluorescence,
h e m a g g lu t in a tio n in h ib it io n an d m e ta b o lic
inhibition arc the m ^ r sensitive tests. Com plem ent
fixation and indirect hem aggiutmation tests are less
sensitive.
The n o n s p e c if ic s e ro lo g ic a l rests
S a ^ m c o K u s M G and cold aggluti nation b a a .T h e
former is done hy m ixing serial dilutions o f the
p a tie n t's u n b e lt e d se ru m Lind a heat k ille d
suspension o f Srreprocaecus M G h and observing
agglutination after overnight incuhation .Lt 3 7 f G.
A title o f 1:2 0 or over is considered suggestive.
T h e c o ld agg lutinatio n test is based o n the
appearance in a high proportion o f cases w ith
primSTy net pi cal pneum o nia, o f m acroglohltlin
antibodies thut agglutinate human group O cells
nt low temperature. T h e patient’s blood sample
should not be refrigerated before reparation o f the
serum, is the agglutinins are readily absorbed by
the homologous erythrocyte? at low temperatures.
Lor til LI rent, serial dilutions o f the patient’s serum
are mixed with an equal volume o f a 0 .2 % washed
h u m a n O g ro u p e ry th ro cy te s, an d f lu m p in g
observed after leavin g at 4 ~C o vern ig h t. T h e
d u m p in g is dissociated at 37 ° C . A litre o f 1 :3 2 or
over is suggestive but demonstration o f rise in litre
in paired serum sam ples is m in e reliable. T h e
indirect Co o m b s test m ay also be positive in some
cases.
Som e strains o f m ycoplasm a frequently isolated
from the urogenital tract of hum an beings and
anim als form very tiny colonics, generally 1 5 - 5 0
pi.i in size. T h e y were called 1 strain or 7 form
mycoplasmas ( T lor tiny),'Flacy arc peculiar in their
ability to hydrolyse urea, w hich is an essential
growth factor m addition to cholesterol. T hi man T
hfra in m vcn p l at; m a r have been re cla ssifie d an
l 'nrapfji.Rrrer uieufynCUlil
G e n ita l infections arc caused by M . bom inis
and Ll urmiytrcurn. T lie y an; trajismitued by sexual
eon tie r, an d m iy cause u re th ritis , p ro ctitis,
balanoposthiti? and Reiter's syndrome in men, and
acute s a lp in g it is , pelvic inflam m atory disease,
ocrvicitis and vaginitis in women. T h e y have also been
associated with infertility akiTtiuru pOstparTum fever,
chutioaiYIrtiortitifi and low birthweigtu o f infant;.
arc
M y c o p la sm a ? tend to cause m ore severe an d
prolonged infections in the I IJ V infected and other
LiumLiEiodcficicnt subjects.
C o n t i n u o u s cell c u ltu re s
m aintained in many laboratories have been found
to be c o n ta m in a te d w ith d iffe re n t sp ecie s o f
C o p y rig h te d m aterial
mycoplasma. The contamination may originate bom
the worker or Irani animal seta or trypsin Used irt
cell c u k u rc . C o n ta m in a tio n generally Jo e s not
produce cytopith.EC effects but may interfere with
the growth of viruses in such Lull cultures and may
also produce misEeading results in serological tests.
Mvcoplasinas growing in cell cultures have- often
hcen m is ta k e n for viruses- Eradication pt
myCPplasolas from infected Cells is difficult.
a rc th e d ru g s o f c h o ic e fo r th e
tre a tm e n t o f m y co p la sm a ] in fe c tio n s . S o m e
u re a p la sm a s are re s is ta n t to t e tra c y c lin e ,
doKvcycJine, the newer macmlide* and quinoloncs.
{ 1 9 3 5 ) found ple u ro p n eu m o n ia-
lik e fo rm s in a c u ltu re o f S trrp ta b a cillu s
iiKjnj'b tom bs and te rated tliem L forms, after Liste r
Institute, Lo udo n, whens the observation was made.
It was subsequently show n that m any bacteria,
e ith e r s p o n ta n e o u s ly or in d u c e d by ce rta in
substances like penicillin, lost part or all o f their
cell wall ana develop into L form s. Such L forms
mav he ‘unstable', when thev revert to their normal
morphology, or 'stable' when they continue in the
cell w all deficient state perm anently. C e ll n a il
deficient forms ( I „ forms, protoplasts, sphcropb-.rs)
may not initiate disease but may be im portant in
bacterial persistence during antibiotic therapy and
subsequent icaLn e n cc o f the infection. It has been
suggested that m ycoplasm ss may represent stable
L form s o f bacteria hut genetic, antigenic and
biochem ical evidence are against the possibility,

Lin J5. 1985. Human mycoplasmal infections. Serologic otacEvation?- .Revinfect Dis 7'216.
Symposium, 1903, The chRiqpng ml* of myouplaainaF in respiratory disease jhh.3AIDS. Clin friifcvt Dis 17;f5uppl.l),
Taylor-RdbinttP [1 and J Etndbuiy 199®, My»fla(ntift dpivutiti, In Topfey M ikI Wsfarft'x jt-Ircrpfcpnfcgy ujii jVfrc-misffJ
Jnfecn'om, 9* edn L:rtdqrt:Artiold

C o p y rig h te d m aterial
 Actinomycetes
Actinom ycetes arc traditionally considered to be
iruiM [ ional forms between bactc ri a and ft] ngi. [ .ike
fungi they form a m ycehil network o f branching
filaments but, tike bacteria, they an: thin, possess
c e ll w alla c o n t a in in g m u ram ie a c id , have
p r o k a r y o tic n u c le i and are s u s c e p tib le to
■ mil bacterial antibiotics. T h e y illre therefore true
bacteria bearing a superficial resemblance to tiingi.
A crm o m yccte s are related to m ycobacteria and
cor ynebacteriiL T h e y arc C ra m positive, nonniorile,
nonsporing, noncapsulated filaments that break up
into bacillary and coccoid dem ents. M o st arc free-
liviiL^, jK L itk u k th in the soil.
ActiflomyceTes include many genera o f medical
in te re st su ch as the a n ae ro b ic A c N n o m yc-e-s,
B ifid o bacterium r Ruthin and aerobic
.\c jc a r J ia , A etin o m a d u ra , D trirnttophU u^ and
S tre p ro Joyces, T h e m a jo r p a th o g e n ic g c n » s
A cfl/io m y ce i is anacrobiic or m kToacrophihc and
nnnucid fast, w hile jV ol-j niia species are aerobic
and may he ad d fast. & >mc sp e d tt o f S tn ^ n w iy ce s
m ay cause disease rarely, but their importance is as
the major source o f antibiotics,
B ollinger ( l£ 7 7 ) found a m ould-like organism in
the lesion Lif'lum py jaw ' (actinomycosis) in cattle.
T h e name actinomyces was coined by I i a n to refer
to the raylike appearance o f the organism in the
granule* that characterise the lesions (Jcrj'rm.rjmncs.
m ean in g ray fu n g u s), W o lf f an d Israel ( 1 8 9 1 )
isolated an anaerobic bacillus from hum an lesions
and produced experimental infection in rabbit:- .md
guinea pigs. T h is was named A rtuM m yw s israelii
It causes human actinomycosis. A ctinom ycosis in
cattle is produced by A . hems.
T h e d isease is a c h r o n ic
granulomatous infection occurring in human beings
anti animals. It is characterised by the development
o f indurated swellings, m ainly in the connective
tissue, suppuration and the discharge o f ‘sulphur
granules'. T h e lesion often points towards the skin,
leading to m ultiple sinuses.
A c t i no m y c o s is in hum an b e in g s is an
endogenous infection. T h e actinomyces species are
norm ally present in the mouth, intestine and vagina
as commensals. T ra u m a foreign bodies or poor oral
Hygiene may favour rissue invasion. A- JStaelii is
the most comm on causative agent- However, other
actinomycetes such as A . riaesAjJtifj'jj A . wscostis,
A . odontoJytkItm, A . meyerr, A . KtrcntHonci and
Propionibactcrium pmpionicum may sometimes he
responsible. Actinom ycosis is usually a co-operative
discare, rhe actinom yces being accom panied by
other associated bacteria, w hich may enhance rhe
pathogenic effect. These include fljfidtafoarleriujTT
Jenfrum, Arrinabacillus aconoinywtemocimrrans,
Eikcnclh convdcns, Haemophilus iphiophilvs,
b ic t e r o id e s , fu so b a cte ria , s ta p h y lo c o c c i an d
anaerobic streptococci.
Actinom ycosis in hum an beings occurs in four
main clinical forms: (1 )o em m lfrcuJw ith indurated
lesions on the cheek and submaxillary regions;
(2) fhofadCi w ith lesions- in the lung that may
involve rhe pleura and pericardium and spread
outwards through the chest xvaU; (3) abdom inal
where ; be lesion is usually around the cecum , w ith
the involvem ent o f the- neighbouring tissues and
C o p y rig h te d m aterial
the; abitnmin j I wall. Sometimes the infection spreads, methods. G e l diffusion and im m unofluorescence
to rhe liver via rhe portal vein. {4) P rh ic. M a n y can differentiate A . isrzelii from other actiuoriycete
c o f pelvic actinm nyem it have keen reported species and from other filamentous anaerobes that
Lit Association with rhe use rtf i ntrautc rin e devices- m av produce granules in tissues.
A c t in o m y c e s have hecn in c r im in a t e d in : ■ : T h e disease occurs throughout
inflam m atory diseases o f the gums (gingivitis and the world bLPt it? incidence in the Advanced countries
periodontitis) and with sublingual p Jiq u e i lending has been d e clin in g probably as a result o f the
ro root GurfaCt curies. A ctin o m y co sis uiav also widespread use o f antibiotics. A ctin o m yco sis is
present ns mycetoma. more com m on in rural v e i l ami In agricultural
Flic diagnosis is made workers. Young male persons ( 10 “ 30 veart old) are
by dem onstrating Actinomycetes in the lesion hy must commonly- affected. T h e reason for th is
m ic ro s c o p y and hy iso la tio n in c u ltu re - T h e predisposition is not know n. A b o ut 60 per cent o f
specim en to be collected is p m . In pulm o nary the Lisei ate Cervicofacial and some 20 per cent
disc. lhc, sputum is collected. ‘Sulphurgrannies may abdominal. Felvir Actinomyces is seen mainly in
L c demonstrated in pus by shaking it up in a test wom en using intrauterine devices.
tube with some saline. O n standing, die granules The disease responds tn prolonged
sedim ent and m a y b e withdrawn with a capillary treatment with penic i Eli n or rctracycli n c .' freatme nt
piperre. G ran u le s may also be obtained by applying will h ive to be continued for several months and
guuae pads over the discharging sinuses. supplemented by surgery where necessary.
I’he granules are white or yellowish and range
in size from minute specks to about 5 mm. Th e )' l To c r.r;i; \.
arc examined m icroscopically under a cover slip. Nocizdia resemble Acriiiomyeetcs morphologically
T h e y arc crushed between elides and stained by bui ate aerobic. All species ate G ra m positive and
G rim stain jtlJ cra m iiLed. T h e gr j iiLile^ Lire, ir1 1ACrt,
bacterial colnnirs ami will he Found to consist ot a
dense network o f thin G ra m positive filam ents,
su rro u n d e d by a p e rip h e ra l ?.one o f sw o lle n
radiating club shaped structure?, presenting a sun
ray appearance. T h e 'clubs" arc b elieved to be
antigen-antibody complexes (Fig. 4 4 .1).
S u lp h u r g ra n u le s or pus c o n t a in in g
actinom ycctcs arc w ashed anil in o cu late d into
[hioglyvollate liquid m edium or streaked on b ra in -
heart infusion agar and incubated anaerobically at
37 LC . In thiogiycollate A , bows products general
turbidity whereas A . isn clii grows as fluffy ball? st
the bottom o f the tube. O n solid media A . ir a e L i
produces small spiders' colonies' in 4 S -7 2 bouts
r!iat becom e heaped Lip, white and irregular or
smooth, large colonies in 1 0 days. O th e r spcdcs
have different types o f colonies.
T h e is o la te is id e n t ifie d bv m ic ro s c o p y ,
, - ■ -r
b io che m ical reaccions and fluorescent antibody i l- - ir »

C o p y rig h te d m aterial
Hidden page
 Miscellaneous Bacteria
Listeria m onocytogenes is a small, coctoid, Gram
positive W.il'lMu-;, with a tOldtaCy to O Q O II \lL chains.
Rough forms may be seen u long filaments. It
exhibits a chfl/adaifcir, slow, tumbling motility'
when grown at 2 5 rC but at 3 7 <3C is nonmmiile.
litis is because pcrifrichous flagella are produced
by the bacillus optimally at 20-30 °C but only
scantily or not at all at 3 7 aC. It is aerobic or
microacrophilic. Grow th is improved when cultures
arc incubated at reduced nsygen tension and with
5-10% COj. It grows best between 30 “C and
3 7 °C, but slow growth occurs even at 4 "C.
Colonies ire hemolytic on blood flgar. L. /MOflO-
cyrogenef ferments glucose, maltose, L rlumnnse
and alpha methyl D-mannosidc, producing at id
without gas. Iris catalase positive. It grows in the
presence of 0,1% potassium tellurite, 10% salt and
at pi I M a n y sernvars have been reengnised.
L . m o n o cy to gen es is w id e ly d istrib u te d in
nature. It has been Isolated from a wide range o f
m a n m u k , binds, fish, ticks and crustacca. 11 occurs
aid el saprophyte in soil, water and sewage. Listeriosis
in hum an beings may present in m any forms. It
m ay cause m e n in g itis o r tneningoencjeplialitisi
particularly in neonates and in the elderly,Infection
o f p reg n an t w om en m ay lead to a b o rtio n or
stillbirth. A sym p to m atic infection o f the tern ale
genital tract m ay cause infertility. Listeriosis inuy
also present as abscesses, conjunctivitis, pharyngitis,
urethritis, pneum onia, infectious m ononucleosis-
like syndrome, endocarditis or septicemia.
M ost hum an infections are caused by scrovar
l /2ia or l /2 h and 4b. Experim ental inoculation m
rabbits causes a m arked m onocytosis {hence the
name m onocytogenes). M onocytosis is a feature o f
hum an listeriosis also. lo n ilk d o n into tlie eyes o f
rabbits produces keratoconjunctivitis y.-In ton test).
H um an infect ion is believed to result from contact
with infected anim als, inhalation of contam inated
dust or ingestion o f contam inated m ilk or food.
O u tb re a k s o f foodborne lis te rio s is have been
known.
L a b o ra to ry d ia g n o sis is e stab lish e d by the
isolation o f the bacillus from appropriate clinical
material su ch as cervical and vaginal secretions,
Inc h it . m e c o n iu m , co rd b lo o d , b lo o d an d
cerebrospinal fluid, Greater success in isolation is
achieved if the m aterials arc stored in tryptose
phosphate or th io g ly co llatc broth at 4 and
subcultures are dim e at weekly internals for 1 - 6
m onths (coM enircfunenfl. Liste rio sis in hum an
beings is being increasingly reported. Isolates are
likely to be missed as nonpathogcrric diphtheroids
u n le ss p ro p e rly in v e s tig a te d . A m p lc illiin ,
c u t r im o iiz o L c a n d g e n ta m ic in arc e ffe ctiv e .
Cephalosporins ate not recommended,
Table 4 > .l lists som e distinguishing features
□ f noELiponngGrain positive bacilli found in clinical
specimens,
Erysipelfjthri.x riitisiopathiti is a slender, nonm otilc,
nonsporiug, noncapsulated G ra m positive rod, w ith
a tendency towards formation o f long filaments. It
is m icroae. o p h ilic on prim ary iso latio n but on
sub cu lm re, grows as an aerobe o r fa cu ltative
■ n. aerobe. It grow s o n o rd in ary m ed ia. IS feck
C o p y rig h te d m aterial
colonics arc developed in tellurite media, It ferments
glucose and lactose, producing acid w ithout gas;
sucrose and m annitol arc not ferine nted. Different
antigenic types have been rccogn isedr
E WnjsEopathjde is a natural parasite of many
an iin .ils. Jr causes Rw:ine erysipelas and hum an
erysipeloid. E turn an infection usually occurs on the
hand or fingers o f persons handling anim als tish
o r a n im a l p ro d u c ts . T h e le sio n s are p a in fu l,
edematous and erythematous, usually involving the
local Lymphnodcs and jo ints. O ccasional eases o f
endocarditis have been reported. T h e bacillus it
sensitive t<> p e n icillin , erythrom ycin and broad
spectrum antibiotics.
T h e nam e Bacterium faecalis tlkaL genes was
originally applied to an ill defined group of G m m
negative hacilli isolated from human fetes-, which
did nor ferment sugars hut produced an alkaline
reaction in litm us m ilk. T h e term A l c a l i g e n c *
ftt c a lit now refers to G r a m n e g a tiv e , s h o rt,
norsporing biu illi, which arc strict aerobes and do
not ferment sugars. T h e y are motile by means of
p tr iir ic h o u s flagella. T h e y ate u su a lly oxidase
positive. N itrate reduction is variable.
A k . /aactEuis a saprophyte found in water and
soil contam inated with decaying organic matter.
T h e y are also commensals in human and animal
intestines. T hey have been isolated from a variety
o f clin ical specimens such as urine, pus and blood.
T h e y have been consid ered responsible fo r a
ty p h o id -lik e fever, u rin a ry infectio ns, infantile
gastroenteritis and suppuration in various parts ot
the body.
Gifttomo&Mcfrrftin? vfeJacrurn lh .l Gram , negative,
nonsporing bacillus, m otile hy means o f polar and
lateral flag e lla. T h e y arc facultative anaerobes
growing on ordinary media and producing violet
pigment soluble in ethanol anti insoluble in water
nnd chloroform . T h e y are oxidase negative and
saprophytic in water and soil. H u m a n infections
haw been recorded m ainly in the tropics and consist
ot skin ledoris with pyem ia and multiple abscesses.
/ ’fevubjL-rerm rn tn cn in go scp tieiim is a G r a m
negative n o nm o tile tod, p ro d u cin g a yellow ish
pigm ent. It is oxidase positive, proteolytic and
weakly temienlulive. It is a ubiquitous saprophyte
capable ot causing o|i|>ortunistic injections. It has
been responsible tor outbreaks o f m eningitis in
newborn infants. Infection in adults leads to a mild
febrile illness.
D o n o v a n ( 1 9 U 5 ] d e sc rib e d th e p re se n ce o f
characteriRtie intracellular bodies in smears from
u lce rate d le sio n s o f a disease now kn o w n as
D o n o v a n o u i. H e considered the bodies to be
parasites. Donovanosis is a venereal disease, first
described by M c L e o d in In d ia in 13 8 2 and seen
m ainly in the tropics. T h e Incubation perit>d ranges
from 1 to 1 2 weeks. It begins as a painless papule
o n the g e n it a lia , w h ic h leads to a s lo w ly
progressive, autoinoculable ulcers. T h e disease runs
a chronic course, Donovan's intracellular bodies
have since been identified as bacteria and named
D M O m iiit grajmJeuTijrjj.,.
Dfegnosifr can be m ade by dem onstration o f
D o n o v a n b o d ies in W r ig h t -G ie m s a sta in e d
impression smears from the lesions. T h e y appear
os rounded a ico h a d lli, 1 - 2 pm, w ithin cystic spaces
in large m ononuclear cells. T h e y show bipolar
condensation o f chromatin, giving a dosed safety
p in appearance in stained smears. Capsules are
usually seen ax dense acidophilic areas around the
bacilli. T h e y arc nonm otile and G ra m negative.
T h e y can Lie grown on egg volk medium and on a
modified Lcvin th al agar, lr is m orphologically and
antigcnically related to klebsiellac,
P a th o g e n ic ity is limited To human heirgs.
lrtraderm al inoculation o f whole cultures or o f an
C o p y rig h te d m aterial
Hidden page
406 * Teulbixik ol Microtiiokigy »
Relapses arc common in untreated c i k i . The
disease can abo occur as outbreaks, in the absence
o f rat hitC- t his co n d itio n first observed in
H av erh ill, U S A , Is called Haverhill fever or
emhema irthricicuni epidemwnn, It ls believed
to be caused also by consumption of raw milk or
water contaminated by rats.
l.lLbnratorydiagnoRiR h bv iRolAtiofi of the bacillus
Iron blood or oilier tjodi fluids, Smears of flue join!
fluid may kIiow pleomorphic Gian] negulive rods.
Aggluiination, C Fard fluorescent antibody tests have
been mjed for serological diagTuiKii;.
${.'■•■"Mum minus Is a short, actively m ot:lc
bacterium, .1-5 * 0.2 -0 .5 ^jm in siix, with two or
three regular spirals and 1-7 amphitrichous flagella.
11 is G ram negative but ls better visualised by
CLemsa or Fontana stains or by dark field
mictoseopy. It was fust observed in a nit hy Carter
il&HH) in India. Japanese workers identified il as
the liiij native agent o f one type o f RBF, ca lied
Suditku, It lias not been culr;-. ated in laboratory'
media.
Spn illaiy R BF bah an intubation period of |—4
weeks. The rat bite wound winch may have healed,
suppurates at the unset of fever, with regional lymph-
ailenopathy. The subsetjnent course is similar to the
streptobacillary type, Mortality rates of up to 10 per
Cent llave bun reported, mainly due h entlocartlitis.
Laboratory diagnosis is bv the microscopic
examination of the blood and exudates from the
lesion, by mtrapcritoncal inoculation into guinea
pigs and mice find by dcmonstisu ion of the spirilla
in their blood art! peritoneal fluid. Biological false
pn-.il ivc reactions for syphilid serology OCCUHS in a
proportion of RJ5K patients, more in the spirillary
form.
Both tv|JeH of RBF respond to penicillin and
tetracyt line. Oral penicillin or doxyevclmc after
rat bite is effective in prophylaxis.
CAMPYLOBACTER
The genus Cam/7vfobrti:tor (Creek, meaning curved
rod) contains slender spirally curved Gram negative
Hods, 0.2-0.5 pm thick and 0.5^5 pm long. They
are- typii .lIH- comma shaped hut crAV occur AS lb"ot
mnltisplral chains. O ld cultures arc coccoid and
pleomorphic. They arc nonsporin# and motile with
a single unsheathed polar flagellum at one or both
poles. G ro w th occurs under m lctfoacrophi 1i C
conditions, 5% oxygen concentration being optimal.
Many pathogenic species are thertnoplrilli:, growing
well at 42 ^C. Cumpvlohacters dn not attack
carbohydrates but arc strongly oxidase positive.
Campylobacters fust gained prominence in the
1970s as a common cause o f human diarrheal
disease, affecting children and adnlts.They can, on
occasion, also cause systemic infections. They are
important veterinary pathogens. CampvlohacteTS of
medical importance arc the following:
Cau-ing diarrheal disease: C- (e/unr, C- coJj,
1;i r:.
Causing extra:utcs’ :n il infection: C\ ferns
Causing abscesses: C. nputorum, i . c*?n,.\'.’.ru.R
C a H PI LOBACTE Ft J h J l NJ
Medically, this is the most im p o rtan t Campylobacter
SpeCiL'S as iL causes .Ul.u k- o f m&Trhea worldwide.
The infection is znnnnti.;, the source being food o f
Animal ohum, especially raw milk. It is part o f tlie
normal intestinal flora nf dom estic animals and
b id s , and is shed in ther.i feces- It can be Isolated
frequently fro m surface waters.
Infection occurs by ingestion. The jejunum and
ileum arc the primary sites of colonisation, but it may
■spread down to the colon and rectum. It is an invasive
pathogen and may involve mesenteric lymph nodes
and CiUSe bacteremia. The incubation period is 1—7
days- The illness starts w :’ b fever, abdominal pam
and warcry diarrhea. Stool contains leucocytes and
blood. The disease is usually self-limited, though
Campylobacter shedding mav com inue for weeks after
recovery Fluid and electrolyte replacement is all tliat
is generally required. W hen needed, erythromycin is
the best anrihiotic.
C o p y rig h te d m aterial
 I .aboratcirv diagnosis depends on isolation of hum ans, a-- v. l-11 as domestic anim als and birds. In
the Campylobacter from feces. Direct microscopic this situ atio n , c lin ic a l disease is in fre q u e n t and
exam ination - phase contrast or dark field usually' confined to children, while older age groups
microscopy to detect the dinting or tum bling arc im m u n e due to subdinical infections.
motility o f the spiral rods, or demonstration o f the The related genus Arcobacteria species ( A
small curved rods in stained smears - may be useful bufi/erj', A . dyaeruphi/a) also caUSe diafrbeal
for presumptive rapid diagnosis. Feces or rectal disease. They are capable o f aerobic growth.
swibs arc plated on selective media. In tase of delay
in c u ltu rin g a transport m edium has to lie
employed. Campylobacters survive for 1-2 weeks This organism was isolated in 191H by Theobald
at J QC in C a r y -Blair transport medium but Smith from infectious abortion in cattle and named
glycerol-saline is not satisfactory. The p ilin g media Vibrio fetus. Ir is a very im portant veterinary
commonly used are Sklrrow’s, Butzler’s or Campy pathogen. Human infectioil by C. fetus may lead
B A P selective media. C. je ju n i, as well ns C coir to bacteremia, sepsis and meningitis.
and C. J.drj', uru thermophilic and dr? not grow at
25 ^C. Inoculated platen are incubated at 42 °C in
an atmosphere o f .?% oxygen, 10% earbon dioxide Spiral, campylnhacter-like bacteria were observed
and H.5% nitrogen. Thermophilic Campylobacters in close apposid-on ro the gascric mucosa in several
can grow well at .17 ° t also hot incubation at higher cases o f gastritis and peptic ulcer, by W nim i and
temperatures suppresses normal fecal flora ro some Marshall in Australia in 1 9 8 3 .They were original Iv
extent. named Camprlobacter pylori. As tliev differed in
Colonies appear usually by 4? hours. They arc many respects from Campylobacters, they have been
nonhemolytic, grey or colourless.* moist, and flat or redesignated as Helicobacter p y lo ri It now appears
convex. Suggest Lve colonies are screened by Gram that hdicobactem have caused human infection from
staining, motility and oxidase tests. Confirmation an cien t tim es. By enaym e im m unoassay,
is by further biochemical tests, including positive hellicohacter antigens have been detected in the
catalase and nitrate reduction tests. intestines of pre-Columbian mummies in the 1?SA.
co/i causes an infection clin ically Tnday, belicnhacters colonise the stomachs nf hnlt
indistinguishable from that due to C-jejuni. ( \ coJr the human population of the world!
is com m only found in healthy pigs, It is Helicobacters inhabit the stomachs of different
differentiated from C . jejuni by the hippuratc animals, each with its own Helicobacter species. II.
hydrolysis test which is positive only in the case of priori is adapted to the human gastric m ucosa.The
C. jejuni, only animal ir infects is the monkey. A larger spiral
f~\ hrj also causes a similar diarrheal disease, lr bacterium o f uncertain fcuofw m y'W . hru'fniaru'* can
can he distinguished from C. jejuni and C. Coh by occasionally infect humun-s and som e anim als like
its resistance to nalidixic acid. cats and dogs also. H , i m z c d i and H . f e n n e l i i a e
C jejuni and C. coli can be serotyped for are assrmiated w ith prootitis in the EIIV infected.
epidemiological purposes.
C. jejuni is the most common bacterial cause of
diarrheal disease in many developed countries - i i. pylori is a t iram negative spiral rod, motile by a
more common than saLmoneOlae or shigdlac. In unipolar tuft of lophotrichous flagella, h grows on
the developing conn cries, C, je ju n i is endemic, chocolate agar or Campylobacter media under
asymptomatic infection being widely prevalent m mlcroaerophiJir conditions* with i-2CAb C O ., and

C o p y rig h te d m aterial
 400 h Textbook tl M crobiology *
p H 6 -7 . A t 37 °C , nulunies lik e 2 -7 days Cu
develop. CoccoiJ forms appear in old cultures. It
produce* oxidase, catalase, phosphatase and E[ \
A distinctive feature is the production o f abundant
unease, and this property lias been Used IS a rapid
diagnostic test in gastric biopsy samples. It does
not metabolic carbohydrates or reduce nitrate.
/ / . priori is global, with a prevalence o f 30-60
per cent— more in the developing than in the
developed countries. The sole source o f II. pylori
is the human gastric mucus. The exact mechanism
of transmission is not clear, hue it is likely to be
oral-oral or fecal—oral. I^overTy,. [Jvercnrwii'.ng and
poor hygiene favour transmission. W ith iiTipraverncnts
in lifestyle, the prevalence ofeliiidhood infecdons has
declined in the developed countries.
After an incubation period o f a few days, H .
pylori causes, in some persons, a mild acute gast: iris
which may last for about two weeks. The ii ifeetion
may he transient in some, but in most, it persists
for years or decades. Such colonisation is usually
asymptomatic, though chronic superficial gastritis
may be demonstrable histologically. The bacteria
are present only in the overlying mucus and do not
iivade the mucosa, Gasl ric antrum is the commonest
site o f colonisation, though any part o f the stomach
may he involved. The infection is strictly confined
to the gastric mucosa, in the stomach, as well as in
areas o f gastric metaplasia and heterotopia in the
duodenum. The exact pathogenic mechanisms are
not clearly understood. Bacterid protease, toxins
or am m onia released by urease activity or
autoimmune responses to gastric antigens may all
contribute.
Pcpr-e ulcer disease occurs in a proportion of
the infected. Chron ic atroph ic gastritis may be seen
in the later stages. The infection is recognised as a
risk factor for gastric m a li^nancies such an
adenocarcinoma and 'mucosa associated lymphoid
tissue ( M A L E ) lymphomas. Such M A LTo pau
appear to he ant igen d riven and are found to regress
after elim ination o f H. pylon by treatment.
Infection induces Ig M , E-dr- Ig A and cellular
immune responses, but they do not seem to be
protec the.
II, pylon shows considerable genetic diversity,
as evident in molecular typing. T h e complete
genome o f the bacterium has been mapped.
Virulence has been associated with certain alleles
in gcnch, such as Gag {cytotmin associated gene)
&nd t^C (vacuolating cytntnxin gene).
T):,agnostic tests aie o f two kinds, invasive and
non invasive. Invasive tests involve cndoscop:c
biopsy o f gastric mucosa, for examination by
microscopy, culture and ureas* tests. Microcopy of
biopsy sections by silver staining or o f G ram
staiitcd smeat' is a useful method. Culture is more
sensitive, but requires expertise and takes 3-7 days.
A bit o f the biopsy material put in a urease indicator
m edium shows positive result in minutes.
Non invasive tests include serology (E L IS A ) and
the 'urease breath test’. In the latter, the subject
drinks a urea solution containing labelled carbon,
which can be detected in the breath. It is sensitive
and reliable, but need* i-rotope assay facilities.
H. pylori is sensitive to several antibiotics and
to bismuth salts. T h e standard treatm en t is a
combination of bismuth subsalicylate, tetracycline
(or amoxycillin) and metronidazole for two weeks.
An alternative schedule employe a proton pump
inhibitor iike omeprazole and clarithrom ycin.
Treatment is indicated only for W, pylori related
gastric or duodenal ulceration and not fo r
asymptomatic colonisation. Drug resistance and
recurrences are frequent.
LEGIONELLA PNEUMOPHILA
The name Legionnaires disease was given to an
apparently new illness which broke out among
members o f the American Legion who attended a
convention '.it Philadelphia :.n 1976. The disease
was characterised by fever, cough and chest pain.
Leading on to pneumonia and often ending fatally.
The causa rive agent has been called Legionella
pneumophila, Subsequent investigations have
revealed that the disease is neither new nor localised.
Copyrighted material
 i Miscellaneous Bacteria ► 409

Infection v.:ih L. pnot/nop/ufa is now known to Lcgioncllac arc widely distributed in natural
. .u:-i ]Tnk'jr. manifestations. I'.Vn <LlS-E11tkjt chi ncal water sources, such as stagnant watery mud and
patterns have been identified and dc-ignatcd as hot springs, where the nutritional and growth
Legionnaires' dismast and Pontiac fever, together requirements for these fastidious bacteria arc
known as fejfjuriei/usjy. provided by some types of algae. Legionellae survive
LegHinnuire’s (listasc cii^iy be either epidemic and multiply inside free-living amebic and other
or sporadic. The incubation period i-- 2 - 1 0 days. protozoa. T h ey also mullipiy in some artificial
T h e (l-scasc presents with fever, nonproductive aquatic environments, which serve as ampliriers.
cough and dyspnea, raj'idly pnigrew ing, if untreated, H um an infection is typically hy inhalation of
to pneumonia. Diarrhea and encephalopathy are aerosols produced by cooling tow ersh air
common, f ase fatality may be 1 5 -2 0 per cent, the Conditioners and shower heads w hich act as
cause o f death bring progressive respiratory failure disseminators. Arrosollsed legionrllae can survive
and shock. All age groups arc susceptible, though for long and can be carried over long distances. No
more cases haw: occurred in the elderly animal reservoir exists, and infection is linf.ied to
Ptjni.'Ui: fever is a milder, non fatal 'mfluenza- human beings. No carrier state is established. M a n -
]ikehillness with fever, chills, myalgia and headache. to-m an transmission does not occur.
Outbreaks with high attack rates may occur. T h e outcom e o f inhalation o f legio n e Mae
T h e discovery ot pneumophila led to the depends on the --lye of the inferring dose, virulence
isolaf ion o f many related hacteri;i, which have been o f the strain and resistance o f the host. Known risk
placed in the genus Legionella, under the family factors arc smoking, alcohol, advanced age,
I jcgionctlaceae. Some 40 species o f leg i ooellae have intercurrent illness, hasp.taUsarion and immuno­
been recognised, many of them w ith multiple deficiency. M e n are more often affected than
semigroups. T h e original isolate in this genus is women. In the developed countries, legionellosis
designated /„ pneumoph/.fa scrogroup 1 (SGlJ, accounts for 1 -3 per cent o f community acquired,
which accounts for nearly all severe infections. and 1CK30 per cent of hospital acquired pneumonias.
Examples o f other species that cause human Its prevalence in the developing countries is not
infection less often are L. micdacJci, L. boz^mnsni. adequately known.
L. d u ift t ft fii and L. j;^r r.1i.i r.'i i. Follow ing entry in to the alveoli through
I-egLnnellae are thin, noncapsulated, hacilii, 2— aerosols, legionellar multiply inside the munuevtrs
5 pm * 0 .3 -0 .1 pm, coccobacillaTy in clinical and macrophages. D issem ination occuts by
materia] and assuming longer forms in culture. endobronchial, hematogenous, lymphatic and
M ost are motile with polar or subpolar flagella, contiguous spread. Because o f then intracellular
T h e y arc G ram negative but stain poorly, location, humoral antibodies arc ineffective.
particularly in smears from clinical specimens. Cellular immunity is responsible for recovery.
They scan better by silver impregnation, but arc Laboratory diagnosis is by rhe demonstration
best visualised by direct fluorescent antibody (D FA ) o f legionellac i n clinical sped me ns, such as sputu m,
staining with monoclonal or polyclonal sera. bronchial aspirate, and lung biopsy, by direct
They have fastidious requirements and grow on fluorescent a n l i b u J y test and culture, by the
complex media such as buffered charcoal, yeast identification nflepnnella antigen*, in urine by latev
CKtraer (B C Y E ) agar, with L-cysteinc and antibiotic agglutination or E L IS A , and hy the detection of
supplements, with 5% C O , at pH 6,9, 35 =C and serum antibody by E L IS A or in d :rcct
90% humidity. Growth is slow and colonics take Lmino nofluorescent assay.
3—6 days to appear For treatm ent, the newer m it r a l idea,

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oxidase po-.itivc, nonmotilch Cram negative rodsf genus \lomxciis. They are part of the normal ora]

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 Rickettsiaceae
Rickettsiae are small, Gram negative bacilli adapted
to obligate intracellular parasitism, and transmitted
by arthropod vectors, They are primary parasites
o f arthropods suxdi . lh lice. flenshticks and mucs, In
which they arc fountL in the alimentary canal. In
vertebrates* including humans, they infect the
vascular endothelium mid reticuloendothelial cells.
The family Rickettsiaccac is named after Howard
Taylor Ricketts who discovered the spotted fever
rickettsia (190b) and died ot typhus fever contracted
during his studies.
T h e fam ily currently comprise-s three
genera— Rickettsia. Grier) ti.i and Ehrlichia which
appear to have descended from a common UUCSlur.
hornier members o f the family, CWeWa burnetii,
which causes Q _fever and Rochalimzca quintjn-.i
causing trench fever have been excluded because
the former is not primarily arthropod-borne and
the latter nut an obligate intracellular parasite, being
capable o f growing in cell tree media, besides being
different in genetic properties.
T h e genus Rickettsia consists of the causative
agents o f rwo groups o f diseases— typhus fevers and
spitted feverc.
In smears from infected tissues,
rickettsiae appear as pleomorphic coccobacilli, 0 .3 -
0.6 (UTl « Q.S^2 pm in size. They are nonmotilc
and noncapsulated. I'hev arc t iram negative, though
they do not take the stain well. They stain bluish
purple with Gicmsa and Castaneda stains and deep
red with Machiavcllo and Gimenez stains.
Under the electron microscope, rickettsiae are
seen to have i three layered cell wall, a trilaminar
plasma membrane and an outer slime layer.,
Rickettsiae are unable to grow ill
cell-tree media. Growth generally occurs in the
cytoplasm o f infected cells hut in the case of the
spotted fever ticket triae, growth may cake place in
the nucleus as well. Rickettsiae grow best in cells
that ate not metabolizing actively. The optimum
temperature tor growth is 32-35 °C.
They art readily cultivated in the yolk sac of
developing chick embrvus, as first shown by C om,
They also grow on mouse fibroblast, I leLa, H E P -
2, Detroit 6 and other continuous cell hues but
Tissue cullures arc not satisfactory for primary
isolation. Laboratory animals such as guinea pigs
anti mice ate useful for the isolation ot rickullsiiie
from patients. They may also he propagated in
arthropods.
R ick ettsiae are readily inactivated
by physical and chemical agents. They are rapidly
destroyed ar 56 °C and at room temperature when
separated from host components, unless preserved
in skim m ed m ilk or a suspending m edium
containing sucrose, potassium phosphate and
glutamate (SPG medium).
Rickcrtsiac arc susceptible to tetracycline,
chloramphenicol anti ciprofloxacin. Penicillin ,irtd
sulphonam ides are ineffective but para-
am m obenwic acid has an inhibiforv action on
ticketcsiae. Sulphonamides may actually enhance
the growth o f rickcttsiac and worsen the condition
if administered to patients.
Rickettsiae have species
and group specific antigens. The immunodominant
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"'Llt face protein antigens (SPA) o f /I. pnotvasreku
and Rr typbi have borh species specific and cross
reactive epitope a, Spotted fever rickettsiae have
dnmmanr outer membrane proteins (O M P ) A and
B, the former being a species specific antigen acting
its an aribe-,in for host I’d]*, and the latter showing
lim ite d cross reaction w ith SPA o f typhus
rkkcttsiac, The third surface antigen is an alkali
stable polysaccharide found in some rickettsiac and
in some strains o f Proteus HadEli, This sharing of
antigens between rickettsiac and proteus. is the haris
for the Wcil-FelLx reaction used for the diagnosis
o f rickettsial infections by the demonstration of
agglutinins Ci) Proteus strains O X O X 2 and
O X K.
Ricketts iae are transmitted to
humans by arthropod vectors through their bite
or feces. O n entry into cite human body, cite
rickettsiac mult ini v locally and enter the blood.
Thev become localised chieflv in rhe vascular
endothelial cells, which enlarge, degenerate and
cause thrombus formation, with partial nr complete
occlusion o f the vascular lum en. T h e overall
pathological features o f the rickettsial diseases arc
similar ami can be explained by the damage to the
vascular endothelium,
'Die long survival of rickettsiac in i .lmous organs
and Lymphatic tissues o f infected men and animals
is a distinctive feature in pathogenesis and is of
importance in rbc epidemiology o f some rickettsial
diseases.
This group o f diseases consists o f epidemic tvphus,
recrudescent typhus [Bril I-Zinsser disease) and
endemic typhus.
(Louseborne typhus,
Classical typhus, Gaol fever) Lias been one of the
great scourge* o f mankind, occurring in devastating
epidemics during rimes o f war and famine, so vividly
described by J Lans Zinsser in his book, 'Rats, Lice
and H istory'. The disease has been reported from
all parts o f the world bur Lias been particularly
common in Russia and Eastern Kunope. Napoleon's
retreat from Moscow was forced by typhus fever
Leaking out among his Troops. During 1^17-1^22,
there were some 25 million caws in Russia, with
about three million deaths. Lenin is said to have
remarked, in reference ro the outbreaks o f louse-
home typhus and relapsing fever rampant during
the Russian revolution, that "either socialism will
defeat the louse nr rhe louse will defeat socialism'!
In recent rimes, rbc main foci have been Eastern
Europe, Africa, South America and Asia. In India,
the endemic spot is Kashmir,
The causative agent o f epidemic typhus is i t
pram zelai named after von Itiowai'jek, who died
o f typhus fever while investigating rhe disease.
Humans ate the only natural vertebrate hosts.
Several animals - guinea pigs, mice, eotton rats
and get hi Is - may be infected experimentally.
Natural infection lu Hying squirrels hat; been
reported from south -eastern LISA. They may
possibly act as reservoir hosts, infection being
spread by the squirrel louse and flea.
The human body louse JVdiefoty- huniddits
corporis is the vector. The head louse may also
transmit the infection but nor the pubic louse. The
lice become infected by feeding on rickettriaemic
patients. The rickettsiac multiply m the gut o f the
lice and appear in the feces in 1-5 days, [je t1
succumb to the infection w ith in 2 -4 weeks,
remaining infective till they die. They can transmit
the infection after about a week o f being infected.
T h e lethal nature o f the infection in the louse
suggests that the association between K. pmwajiefcjj
and its vector is relatively recent and not well
established. Lice may lie transferred from person to
person. Being sensitive tn temperature changes in the
host, they leave the febrile patient or the cooling carcass
and parasitise other persons. Lice defecate while
feeding. Infect:on retransmitted when the contaminated
louse feces is tubbed through the minute abrasions
caused by scratching- Occasionally, infection may also
be transmitted by aerosols o f dried louse feces through
inhalation or through the conjunctiva,
C o p y rig h te d m ateri
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ricks, which therefore act aa both vectors and
reservoirs. T h e infection may be transmitted to
vertebrate hosts by any of the larval stages or by
adult ticks. Ticks arc not harmed hy the rickettsiae
and remain ir letted fur life. The rickettsiae art shed
in tick fcccs but transmission lo human beings is
primarily by bite, as the ricketLsiae also invade the
salivary glands of the Neks. AJ] riche r[Scifi o f [Jus.
group pass through natural cycles in domestic and
wild, animals or birds.
Rocky MoirnraiJi sported fever is the most
serious type o f sported fewr and is the First to have
been described- It is prevalent in marry parts of North
■nuE South A m erica and is transm it ted by
Dermaeenfor Andcn w ii and related species o f ricks.
T ick typhus in several parts o f Europe* Africa
and Asia ]£ caused by R . conori, strains o f which
isolated, from the Mediterranean littoral, Kenya,
South Adrien and India are indistinguishable. The
species is named after Conor, who provided rhe
first description o f the Mediterranean disease ‘fievre
boutonncuac' (1910). The disease was first observed
in India by M eglW (1^17) in the foothills ot the
Himalayas.The investigation (it Kalra, Ran, Soman,
Helig and Naidu had established that the disease
is found it] many parts o f In d ia. T h e rick
RhipKCptwhl* .sj ngurneu* is the most important
vector. Hzcmaphysafo leachi. AmNyowma anti
Hyalom m ii ticks can also transmit the infection.
The mildest rickettsial disease
o f humans is a self-lim ited, nonfaral, vesicular
eMnthem first observed in New York (1^46). The
name is derived from rhe resemblance o f the disease
to chic ken pox. It is also called vesicular or
YniceUrform rickcrrsiosis. The causative agent is
R. iikitrj (From akari, meaning mite). The reservoir
o f infection ls the domestic mouse, M uj muscutus
and the vector i,s the mite, Liponyssoides (Jornuerfy
Aliodentimyssus) ju ^ u in riu d n which transovarinl
Transmission occurs. R. akari ba> also been isolated
trotn wild rodents in Korea. The disease has also
been reported from Eastern Europe and Korea.
Scrub typhus is caused by Orient!a tsutsugamushi
[formerly R, tsufs^gatnush/. R. one/j fairs), It occurs
all along east Asia, from Korea to Indonesia* and
in the Pacific Islands including Australia* I t was
first observed in Japan where ir was found to be
Transmitted bv mites. The disease was thereto re
called rsutetfgirmwhi (from fsorsug-fl* meaning
dangerous, and itiushi meaning insect nr mite). It
is a place disease and is found only in areas with a
smtiahk- climate* plenty ot moisture and scrub
vegetation. T h e vectors are trum biculid mites
belonging to rhe germs LeptotronihidiuTn— L,
akiimushr in Japan and L dehensis in. India. The
mites inhabit sharply demarcated areas in the sod
where the microecosystem iu favourable (mire
isl.miis). Human, beings lire in f e c t e d w h e n they
tre s p a s s into th e s e m it e islands a n d are h it t e n hy
th e m ile la r v a e (rJirggerr). The m it e fe e d s o n the
strum o f warm blooded animals only once during
its cycle o f development* and aduli miles- feed only
on p l a n t s . T h e m icruhes are t r a n s m i t t e d
jnmsovarially in mites, Various rodents arid birds
acr ils reservoirs and also help in spreading th e
o r it n t ia c t o fre s h areas.
Scrub typhus, originally found in scrub jungles
IliS also been identified in a variety of other habitats,
such as sandy beaches, mountain deserts and
equatorial rain forests. The term r h ig g e r - b o m e
typhus has therefore been suggested is a more apt
designation. Four factors are essential for the
establishment n f a microfocus o f infection* viz.,
coexistence and intimate relationship among O.
rsursugafnt^fii, chiggers* rats and secondary or
transitional forms o f vegetation (known as the
xoufioqc tetrad),
The incubation period is 1-3 weeks. Patients
typically develop a characteristic eschar at the utc
of the mile bite, with regional tymphadenopathv
and a macufopapular rash* The disease sere in with
fever, headache and Conjunctival injection,
C o p y rig h te d m aterial
Encephalitis arid pneumonia may be seen in severe
cases. The disease is not a serious problem in civilian
practice but assumes ^te.U importance in military
medicine, especially during jungle warfare, as was
recognised in the In do-Burmese theatre in the
Second World War.
Considerable differences atist among different
strains o f O. rsutf L^'-Jinusitoj in antigenic properties
and virulence, a factor that complicates acrodiagnosls
and iinmunoprophylajciH. Three major antigenic
types have been recognised— Karp, Gilliam and
Karo,
arc small, Gram negative, obligate]}'
mrraedluar bacteria which have an afti n in1towards
blood cells, In the cytoplasm of infected phagocytic
cells, they grow within phagosomes as mulbcrry-
Eike clusters called morula (morula, meaning
mulberry). Tbev are dek-bome. Similar organisms
under the names o f Awtplazm?, C o W n V and
N eo rick ctisia , had long been known to veterinary
scieniis Es an etulugicil jaunts of hckburtle intcctu ■ils
□f cattle and sheep. Three human infections caused
by [ltd group ol'organism's have been identified.
The first o f these human diseases, reported from
Japan in 1954, was a case resembling glandular
fever who showed serological response against the
agent o f canine ehrlichiosis. The etiological agent
lias b*ren named Etiflichi* sennets* (from 'scrmetsu',
the Japanese word for glandular fever). It is endemic
in Japan and parts o f S w th Fast Asia. It causes
lymphoid hyperplasia and atypical lymphocytosis.
N o arthropod vector has been identified. Human
infection is suspected to be caused by ingestion of
fish earning infected flukes.
T h e second type o f infer [ion is 'human
monocytic ehrlichiosis1 caused by E. cfiaffeeniis.
It is transmitted by Armblynmma ticks, Deer and
rodents are believed t<■be reservoir hosts. Human
disease is associated with feucopetlia, thrombo­
cytopenia and elevated liver enzymes. Multisystem
involvement and fatality may occur.
T h e third is 'human granulocytic ehrlichiosis'
caused by an organism either identical with or
closely related to the equine pathogen, E. ctflii
(probably £. phagfxyrophiia). It is transmitted by
bcodcs ticks. Dccr, cattle and sheep are the suspected
reservoir. Leucopenia and thrombocytopenia aro
seen in ]tatLen,&.. Gietnsa staimed blood films may
show morula form o f the ehrlichia.
Dmcycyclirte is recommended tor treatment of
ehrlichioses.
R ickettsial diseases
may be diagnosed in the laboratory either by
isolation o f the rickettsiae or by serology. As
rickcttsiac are highly Infectious and have caused
several ^-riousand fatal infections among laboratory
workers, their isolation should be attempted with
utmost care and only in laboratories equipped with
appropriate safety provisions.
Rickettsiae maybe isolated in male guinea pigs
or mice from patients in the early phase o f the
disease. Blood clot ground in skimmed milk or any
suitable suspending m edium is inoculated
intTui'CMtonvidlv. The animals have to he observed
fer 3 -4 weeks and rheir temperature recorded daily
T h e ir response to rickettsial infection varies, hi
Kuckv Mountain spotted fever, guinea pigs develop
fever, scrotal necrosis and may even die. W irh R,
ivphi, R. conofi and R. akan. they develop fever
and tunica reaction. R. prowazekii produces only
fever without any testicular inflammation. Smears
from the peritoneum, tunica and spleen ot infected
animals may be stained by Giemsa or Ginvent,
methods ro demonstrate the rickettriae-
Though laboratory strains o f rickettsiae grow
profusely in the yolk sac o f chick embryos, this
method and tissue culture are not suitable for
primary isolation. Egg and animal inoculation
methods have been replaced by the faster and more
sensitive cell cultures. Rickettsiae grow well in 3
to 5 days on YeroccII M R C 5 cell cover slip
cultures and can be id en tifie d by im m u n o ­
fluorescence using group and strain specific
monoclonal antibodies.
C o p y rig h te d m aterial
 Serohigicat diagnosis may he by tfce hcrerophile
W ei I-F e lix reaction or hy specific tcsrs using
rickettsial antigens, The W cil-Felix reaction lb an
dggluri n it inn itrsc in which sera are tested tor
agglutinins to the O antigens o f certain nomnoriJe
Proteus strains 0 \ l 9 hO X 2 and O X K. The test
was developed from the chance observation of Weil
and Felix (1 916) that a Ptoteus Htmin isolated from
the iitire o f a patient of epidemic typhus wan
agglutinated by the patient's semm as well as by
tI ll- sera o f other typhus patients. Tile basis o f die
test is the sharing of an alkali'Stable carbohydrate
antigen by some lickettsiae and by certain strains
o f Proteus, P. vu^ahr O X 19, and O X 2 and P
mirabilisOX K .T h c test is usually dune an a tube
agglutination, though rapid slide agglutination
methods have been employed for screening.
Sera from epidem ic and endemic typhus
aggluiiditie O X 19 and sometimes O X 2 also. The
tent is negative nr only weakly positive in llrill-
Ziusscr disease. In rickbornc spotted fever both O X
l 4^ and O X 2 are agglutinated. OX K agglutinins
are found only in scrub typhus. The test is negative
in rickettsial poxr trench tever, and QJtVCT (Table
96.2).
The W cil-Feiix reaction is a simple nnd useful
te^t tnr rhe diagnosis of some ricketttiul diseases.
The antibody appear* rapidly during the course Mt
the disease, reaches peak titres o f upto 1.1000 or
1:5000 bv the second week and declines rapidly
during COOValCKCflCe. False pOfltiVt reaction may
occur in Home cases of urinary nr other infections
Epidemic Typhus + -h +
H rill ?imsrr diiic-ase Usually negative or (week pr^ti™
Endemic typhus 4 +■•
Ttckbormc spotted fever 4- +■
Scrub typhus - -
by Prorcus and at times in typhoid fever and liver
diseases. I fence it is desirable to demonstrate a rise
in titre o f antibodies for the diagnosis o f rickettsial
infection,
T h e most frequently used serological method
using rickettsial antigens is the complement fixation
rest. This may be done using the group specific
soluble antigen, or the type specific washed
rickettsial antigen. The former test is in routine
use bur the latter is n e c tu u y fbr differentiation
between epidemic and endemic typhus. Other
s e r o lo g ic a l te a rs in c lu d e a g g lu t in a t io n o f r ic k e tt s ia l
suspensions, p-issivc hemagglutination o f red cells
sensitised by E 3 5 {erythrocyte sensitising
substance), rosin neutralisation, imtTiuno-
fbomccnce am] radioisorope precipitation.
Rickettsia] diseases may
be prevented by general measures such as control
o f vectors arid animal reservoirs. Immunisation is
useful in special situations. Killed am! live vaccines
have been prepared against epidemic typhus. "Hie
earliest <if these was phenolised intestinal contents
o f hce infected per rectum with R. fu-owzzvkii
(W q g fs vaccine). This was loci complicated for
mass production. Castaneda developed a
fo rm a l!iiiscd mouse lung vaccine. E ffective
vaccination hci'iLmc possihlc only after Cox
developed the inactivated yolk sac vaccine, A live
vaccine using the attenuated smiin Elias been found
to be highly immunogenic hut a proportion of
vaccinecs develop m ild disease. T h e C o * type
vaccine has also been prepared again** Rocky
* -
-
± -
+■ + -
+ + +
C o p y rig h te d m aterial
M aimt-un spurted fever. However, there is- no
satisfactory vaccine available against any o f the
rickettsial diseases
D e rric k (1 9 3 5 ) investg’a ting an outbreak o f
Cyphuid-like lever in abattoir workers in Brisbane,
Australia, transmitted the infection Co guinea pigs
by inoculation of blood from patients. As the
etiological agent o f the disease wan unknown, it
was referred to as 'Query' or fever. As Burnet
identified the causative agent as a riuketrsid, it w ;lh
named R. (mnierii. A t about the same time, the
same agent was isolated independently by Cox in
the U SA from ticks, and was called K, diaporiea,
the name referring to its ability to pass through
pores o f filters impermeable to other riekettslae.
Thu A ustralian and A m erican strains were
subsequently shown to be identical. As the Q jever
agent differed from orher rickcttsLae in many
ieatures (smaller s i/jt gTcator resistance to licit and
drying, major transmit or route being inhalation
or ingestion, idependem o f arthropod vectors), it
has been separated from rickettsiac into a special
genus and renamed Caxiclla burnetii. It has been
assigned to the group Pmtobucteriii along wi th other
genera like Lcgionclh and Fmncisdla
Q^fever is distributed worldwide, is a zoonosis
solidly established in domestic livestock. W ild
animals such as the bandicoot may be the primary
reservoir. It is transmitted among them, and to cattle,
sheep and poultry by ixodid ticks. TtansovariaL
transmission occurs in ricks. Coxiclla arc abundant
in riuk feces and survive in dried feces for Long
periods. They are shed in the m ilk o f infected
animals. They are particularly abundant in their
products o f conception and contam inate the
environment at parturition..
H 13man infection may occur occupationally
through handling wool or hides, meat or other
animal products contaminated with tire organism.
Drinking infected milk can transmit the infection-
Coxiella may enter through abraded skin, mucosa.
Itlngs or intestinal tract. PefSOn^tO-peMOn
transmission is a rarity. Ticks do not seem to be
Important in human infection,
Cox. burjierij is widely prevalent in birds and
animals in Tndia, as shown hi- serological surveys,
hut human disease has been identified only rarely.
The human disease is an acute systemic infection
characterised by nn interstitial pneumonia. The
clinical picture is very variable and asymptomatic
infections very common. In chromic Q_feve, the
coxiclla spreads through almost all organs and may
cause hepatitis;, m eningoencephalitis or
endocarditis. Spontaneous recovery is usual. The
coxiclla may remain latent ill the tissues o f patients
for 2-3 years.
Cox. burner ji is an obligate intracellular
pathogen, prim arily infecting the monocytc-
niacrophage cells. It occurs as rods G.2-Q.4 pm x
0.4-1.0 pm nr as spheres 0-3-0.4 pm in diametuT.
It is filterable. G enerally regarded as G ram
negative, A it is better stained with Gimincz and
other rickettsiac strains
In dried feces or wool it survives tisi a year or
mure at 4 'G and in meat at least for one month. It
is not completely inactivated at 60 °C or by l':kfr
phenol in one hour. In m ilk it may survive
pasteurisation hi' the holding method, hut the flush
method is effective, (t grows well in the Volk SAC o f
chick embryos and in various cell cultures.
Cox. bit m et ii shows phase variation, fresh
isolates arc in Phase 1. k becomes Phase II on
repeated passage in yolk sac, but reverts to Phase I
by passaging in guinea pig*- Phase 1 cells are
autoagglutinablc and are phaguCylOStd ill the
absence o f antibody. Phase I activity is due to n
periudate-se]is-itive trichloracetic acid-soluble
surface carbohydrate. Phase 1 is a more powerful
immv nogen than Phase II and elhits- gm >d antilvjdy
response to both L and I I antigens. Phase 11 antigen
is more suitable for complement fixation tests. Q_
fever sera do not cross-react with rickettsial or
proteus bacillus antigens.
Laboratory diagnosis is by serology, by
C o p y rig h te d m aterial
com plem ent r'ix:iTion or indirect im m u n o ­
fluorescence assay- Illa tio n of the coxiclla from
Wood, sputum or other clinical spC^itucil^ IS
possible, but is not recommended due to the hazard
ot laboratory infection.
Vaccines have been prepared from tormsiHin
killed whole cells, trichloracetic acid extracts and
,1 tte minted strains, hut th.ev are not Jnr general use.
Treatment is w ith doxycycline. lit endocarditis
prolonged treatm ent tv irk com hi nations [if
tetracycline, cotrimoxazolc or rifampicin may be
required.
Ihirronellac are tiny Gram negative bacilli, usually
tran sm itted by arthropods, which invade
manimaEian endothelial cells and blood cells.
Human pathogenic strains are B. btaltifonais,
11. quin tin a and B. henselae. The genus contains
species causing a number o f tickbome fevers or'
antmah. Identification acid classification of member,
o f bartnucllac. rickemioe, chlamydia* and related
bacteria often depend on sophisticated molecular
methods tike lb? R N A analysis.
In 1870, when the railway line from Lima to Oroya
in Peru was being built, an outbreak o f fever killed
lhuusands o f workmen. T h e disease was called
Omys fever, which wan seen in the mountainous
parts of Ptm , Columbia anti Ecuador in Sooth
America. Some o f the survivors developed nodular
ulcerating skin lesions, called vetn^a peruana. The
common etiology o f these two conditions was
established tragically in IKS5 by the Peruvian
medical student Daniel Carrion. H e inoculated
himself with material from verruga and developed
Oroya fever from which he died, Oroya fever is
therefore also known as Carrion's disease.
Oroya fever presents as fever and progressive
anemia due to bacterial invasion of erythrocytes.
M ortality is high in untreated eases. A late sequel
in survivors or in those with asymptomatic infection
is verruga peruana. H. badSHormin is seen inside
erythrocytes and in rh t skin lesions. It is a
plcomorphoic Gram negative rod, which is motile
bv a tuft ot polar flagella. It can he cultivated in
semi solid agar with rahhit nr human hlnnd.
During the E'irst World War, over a million cases
o f a disease known as french f e w or Sve-day fever
occurred among soldiers fighting in the trenches
in Europe. T h e disease was not fatal buc because of
its slow course and prolonged convalescence, it
caused very considerable loss of manpower.
Trench fever is an exclusively human disease
and no animal reservoir is known. It is transmitted
hr the body louse. T h e Itces u-f lice becomes
infectious 5 -1 0 days after an infectious meal. The
lice arc unharmed and remain infective throughout
their live^-. Vertical trailsmissinn dit>es not occur in.
lice. The causative agent was identified as a rickettsia
and named R. quintanj {from quintan-.!, meaning
fifth, referring to 'five-day fever', a synonym for
trench fever), As it was found to differ from
ticket! sue in a number o f respects, including its
ability' to grow in cell-tree culture media such as
blood agar, it was separated into a new genus
Roclualim.LC.L (after [la Rocha L im a, an carlv
investigator ot rickettsial diseases), In a subsequent
taxo nominal shift, it has been reclassified as
Bartonella and named B. quintna.
The disease frequently leads to ;l chronic or
latent infection. Recrudescence may occur its in
H rill-/in s s c r disease and relapses have been
reported as long as 20 veins after die primary ducaK.
The chronic infection anti late relapses help to
maintuin the hartoncEJa in the absence of animal
reservoirs.
french fever was thought to have vanished with
the world wars. But isolation o f IS. qtu'jirana from
Tunisia and Mexico recently suggests that the
C o p y rig h te d m aterial
 i R:cliell&iaceae ► 421

disease may be more widely di-tributed i|\m is
realised. Trench fever cases have beer iident:lied in
some homeless persons liv in g ill unsanitary
conditions in the USA.
BARTONELLA HENSELAE
A febrile illness with lymphadeno-pathy following
a cat scratch had been known for long under chc
name 'ear scratch disease', but its etiology rcm.ii net)
elusive. R. henselae has heen isolated from the blood
o f parients, in blood media after prolonged
illfo b u io r and js now cnnsMerrd as its etiological
agent. It can be demonstrated in lymph node biopsy
smears and sections by Waithiir-SratTy st.iining-
B. heiiseiac has been also linked with two other
conditions, seen more commonly in 1 11V infected
and other immunode fie lent persons. These ate
baciUary angiomatous, in which vascular nodules
or Turnouts appear on the s.-on* mucosa and other
locations, and bacillary pcliositi involving the Liver
and spleen.
Angiomatosis may also be due to B, quj'rrttina in
some cases.- Another organism Afipia felt* had also
been proposed as a cause o f cat scratch disease.

F u rth e r Reading
Adal KA. J995. Eiarfnnclla: N*w spciitu ami new disease, fiev .Wed MfcnjifioJ&: 155.
B juj OG and D Paretsky 1983. Qfever and OwwJfci tarmc/ji. M i c n i b i f t l R n 47:127.
H ul[h ! Pci j L. 1995. Uurtun granulckyne ehrlichiosis. Lancer 34b: 782.
1'lshbrin [IB ct al l^9J. Human I'.l'.rI.i hinsih. . ir ;11 Jii! .t Jo/ I 2D
Kochlar JU vt nl. 199-t. ^fuchdJrmie-j henselae infecrinn. / A m M e d Assoc 271:531.
Mmirin ^1 and I I li 11 199*?. Q_levirf. O ij) ,Vf.i rtJi':j.iil ilfV 12:5 18.
Haoulr D :md V Rem* 1 99V. Rkkiensoses Jn paradigms of new nr emerging infecti jus diseases. C lin AlicrohjoJ fiev 10:6-94.
St/hull"* 5,1G , 196R. A L -ituiv n f tvarimbcllnM- (C jo ions disease). A n tJ T h y 1 M e d 17:503.
Walker |)H ind JS 13nmli.-r !9%. Emergence nt" Ehrlit hi-osis as a human health problem, t'merpjyr JnirnT /7js 2:N<A.
Wnudwarri TE, 197.1. A hitter kid HCMHt njf riekL-nsLal diseases with a discussion of the inched ptMttOt, J Inf'Di*
127:585.
Yrinswr PI. 1915. R it \, Lw e m d Siismn'. London: George, R-outledge and Sons Ltd.

C o p y rig h te d m aterial
 Chlamydiae
Chlamydiae are obligarc intracellular bacterial
p u u ile j 0 ^IniMUU) animals and birds with tropififn
for squamous epithelial celts and macrophages ot
the respirator)' and gastrointestinal tracts. Due ns
their filterabilitv and [allure to grow in cell-free
media, they Were considered to he viruses. Rased
on ihe human diseases they were then known rn
cause, they were called psittacosis-
lym phogranulom a-irachoim (P LT1 viruses, or
noncommittally ns ’ P IT agents'. However, they
difter from viruses in many respects. They' possess
both D N A and R N A , have cl-LL walls anti
ribosomes, replicate by biliary fission without an
‘eclipse phase', and are susceptible to the usual
antibiotics and chemotherapeutic agrnts, "J'hcy are
Therefore accepted ,is bacteria. Unlike other bacteria,
they do not have peptidoglycan cell walls. They
lack enzymes o f rhv electron transport chain and
so require A T P and mitrienr resources from host
cells. They have therefore been called enerjyr
parasites.
In recognition o f the pioneering studies o f Sir
Samuel Ikdson on psittacosis, the name B c t is o n i x
was proposed for this group. However, they are
now officially classified as bacteria belonging to
the genus Cklamydiaf in the family Chlamydiaecae,
under the rnder Chkmydiales.
Tkc genus C htsunydin contains four species:
— C trachomatis, C. psittad, C\pneumoniae, which
can affect human b; and the fourth species, C.
peeorum created recently to include some Strains
affecting ruminants. Species differentiation is based
on growth characters, nucleic acid profile, antigens,
plasmids and nature of the inclusion body.
C, rmchomatis strains form compact inclusions
w ith the g ly co g e n m a trix , are sen sitiv e to
mlphonamidcs, and are natural parasites o f humans,
usually causing haralised infections o f the eyes and
genitals. £7. psittad strains form diffuse vacuolated
idelusions wi [hoot rh e glycogen matrix, a re resi stan t
to sulphonamidcs and arc natural parasites o f birds
and animals, capable o f causing pneumonia and
generalised infections in humans. C. pneumonixe
is ,tn exclusive human pathogen with no animal or
avian host. It is a common cause of acute respiratory
disease worldwide.
Chlamydiae occur in two forms, the elementary
bod}- and tlic reticulate body (formerly also called
[fie 'initial iiody'). The elementary body is the
extracellular, infective form. It is a spherical particle,
200-300 nrn in diameter, with u righl rtibm inar
cell wall similar to the cell walls o f C„iram negative
bacteria, and an electron dense nucleoid. T h e
reticulate body is the intracellular growing and
replicative form, 500—10Q0 rim in si™. Its cell wall
is fragile and pliable, leading to pleomorphigm.,
Infection is initiated by the attachment o f the
elementary body to the surface o f a. susceptible
epithelial cell, followed bv its entiotytnsis (Fig.
47.Ij. Inside the host cell, the elementary body Lies
within [he endosome, lie log separated frnm the host
cell cytoplasm hy the cndosomal membrane
throughout its active growth eyde. By about eight
hours, the elementary hodv within the endwome
undergoes spheroplast-likc transformation to the
large reticulate body, which begins to divide by
binary fission by 12 hours. By 2 0 -2 4 hours, the
C o p y rig h te d m aterial
 I. EIcnKiitiry b«ty fEB}: 2. EB riUchc* (is l^3I raxpinr, J. ] EittM t LtU b y eHkKYHMLS, B y S \nmni 4. E B i c o n u i K d
Iniih ftiiiddit (RB); 5. FLoh cell powtfi m fle d try 12 hour*. I mderpoi^g finiM i: 6 . IHy 34 hours, InHuutn body with SB
ind tlevelofniq. EB; 7. B-y 3thhniin* incluutfii tody conuiiUB

i.%. 47J ’ppodooflvi cy®te til GM^mydi

pleomorphic progeny show central condensation During the active intracellular growth o f the
and arc converted lu elementary bodies. Binary organism , the chlam ydia specific Lipopoly-
fission continues till about 40 hours. The saccharides accumulate on the host cell surface.
developing chlamydial microcolony within llu:hOxi T h is h ig h ly antigen i. m areri&I induces
ceil is called (he in d u fio n body. T h e mature inflam maroy and immunological responses which
inclusion body caiitains L011 SCO elem ental bodies contribute [u the pathogenesis chlamydial
which arc ultimately released from the host cell. diseases.
In C. p&ittad infections, the h w l td l is severely thlam vdiiic can he propagated in [he mouse,
damaged and release of the d c m m tiry bodies chick embrvo or in cell culture though, they show
occurs within 48 hours by hc^r cell lyeia. W irh £ '■ individual variations in susceptibility.
foaehomatJS^ the [nature iodusion LpiiC£i - to he isrifltsmie: Chlatnydiac are heat Labile, heing
cxocctoscd in 7 2 ^96 hours, rhe host cdl being left inactivated within minutes at 56 :'C , They arc
with a scar (Fig- 47rl). susceptible to ethanol, ether and low concentrations

C o p y rig h te d m aterial
 C. trachomatis A, B, Hi, C
C. trachornabs Q ,F. F ,G ,H nI.J, K
C trachomatis L l, IJ±, L3
C. psjtJacj M m v serotypes
C, pneumoniae Only one serotype
■I"TULLh.i i linJ.I iH iyp*» M flfinffrf with I he disease
o f phcnLi] and formalin, Infectivicy is maintained
for several days at J "C. They cm be preserved
&O tni at -7 0 "C nr Impbi lined.
Chlamydia*: potters
three major kinds o f antigens, T he first is the heat
stable, genus specific antigen common to all
ell bunpdUK- Thin ls a lipopolysscthlridc rese milling
the IP S H>r' enteric Cram negative bacilli. This is
present in all stages o f the developmental cycle and
ran he identified by the complement fixation test.
The second type ol antigens are lllc specie*-HjiccLfic
protcin antigens present at the envelope tu rfu t.
These arc present in all strains o f a chlamydial
species. They help in classifying chlamydiac into
the Species— trjiTlonnirji, pSjrtflCi, pneunumiae jm i
ptcorum . The third hind ot antigens help in
intmepedes typing, as they ure found only in sonic
members o f a species. They arc located on the major
outer membrane proteins (M O M P ) and can be
demonstrated bv microirrmainofluoiesccncc. bv
m icro-IF. chlamydiac have been classified inic
many serological variants (serovara, serotypes).
C- m c /io m a tu is classified into two. broad
fuovars (biological variants) which cause trachoma,
inclusion conjunctivitis (T R .IC ) and
lymphogranuloma venereum (L G V ), respectively
T h e T R IG biovar has been classified into 12
serovars -\, H, Ha and C causing blinding
trachoma in endemic areas, and serovars 1? to K
associated with the less serious nccular infection,
inclusion conjunctivitis and with various genital
infections, Setwars L l, L2 and L3 cause L G V and
Lndemic blinding trachoma
Inclusion ci>njunctivitu (neonatal and adult)
Genital chiamdiasis
Intani pneumonia
Lymphogranuloma venereum
Psittacosis
Acute respiratory disease
hemorrhagic proctitis.
The serological classification o f L\ pirittan in
COinpleA, itiaiLV Serotypes having been identified-
C- pneumoniae has not been subdas&ificd as only
one serotype is known (Table 47.1).
Four approaches are
available for laboratory diagnosis o f chlamydial
infections: (1) microscopic demonstration of
in el us urn or elementary bodies. (2) isolation o f
chlamydia; f l) demonstration of chlamydial antigen
and (4) dem onstration o f antibodies or
hyperseii si tivi ty.
Chlamydial elementary bodies and inclusions
ire large enough ro he seen under the light
microscope. Chlamydia are G iam negative but are
Stained better bv Gienisa. Castaneda, Machiavcllo
or G i mines stains. Microscopic examination o f
Gicmsa stained conjunctival scrapings for the
inclusion bodies is useful in the diagnosis of ocular
infections particularly in neonatal inclusion
conjunctivitis, Because o f the glycogen matrix o f
£’. trachomatis inclusions, they can be stained with
Lugot's iodine. Iodine staining of conjunctival
scrapings has been used as a rapid and simple
screening method for trachoma and inclusion
conjunctivitis. However, its sensitivity is poor as
iodine staining occurs only in certain stages of
development o f the inclusions. It is. however, useful
in the rapid screening for chlamydial inclusions in
cell cultures inoculated with cLini-cal samples. Iodine
staining is not applicable in C. pshtma because its
inclusions do rot contain glycogen.
C o p y rig h te d m aterial
Hidden page
or type-specific m ic ro -]R The C\? rest is used
m ainly in invasive chlam ydial in fe rn o ns—
psittacosis and L G V . A fourfold rise in titles is
diagnostic- As low titre, grnup-spccific .mtibody
may be present in the sera o f many persons due to
exposure to other chlamydia^ a single C F antibody
lest is not diagnostic of psittacosis nr L G V unless
rhe ritn: is high - 1:64 or greater. C P test is of
tittle value lii T R IG infections, in which micro- IF
is more usefiih M icro-] F can test IgG and IgM
nntihodvseparately. Litres nfLS or greater arc usual
in infected persons. Enzyme iirrrnimoassays also arc
iv ili table.The initial antibody response is IgM ,
which is replaced by Ig G utter about a month.
Recurrent infection with the same serotype induces
only IgG response. As low tirre antibodies are
frequently seen in healthy individu al the diagnostic
criteria for serology are seruconvcrviun, fourfold
rise in IgG litre or presence o f Ig M antibody. High
litre antibodies arc usually seen only in infant
pneumonia, sniping!ii* and L G V .
Demi mstratiou of bypersensi tivLly by skin tes ti ng
(Fnci's test)w.ts widely used formerly for diagnosis
o f LG V but has been given up because false positive
results arc very frequent.
C, tmehamatia is a leading cause o f ocular and
genital infections worldwide,
Trachoma is n, chronic keratocon­
junctivitis characterised by follicular hypertrophy
papillary hyperplasia, panrus formation ;md in rhe
[ate stages, cicatrisation. The name trachoma is
derived from the G reet riukiius [rough] referring
to the roughness of the conjunctiva in the disease.
Though HaLberstaedter and Prownzelt in 190"
transm itted the infection to orangutans and
dem onstrated in conjunctival smears the
characteristic inclusion body that iiears then names,
cultivation of the chlamydia became possible only
htilf a century later, when Tang and colleagues.
(1957) grew it in the yolk sac o f eggs.
Infection is transmitted from eye*to-cvc by
fingers or fornitea. Flies may Transmit the intection
mechanically. It may also be carried by dust, in
which case infection may he facilitated by minor
abrasions caused by dust particles. The incubation
period is variable and influenced by the dose ol
infection. Onset is insidious.
Tr.ichoma ha*. been classified into several StiigCS-
Thc earliest is trachoma dubium, where the disease
is ulsL a suspicion. Protrachoma is the stage of
conjunctival Lesion before follicles become visible.
The inclusion bodies are nor usually demonstrable
in these carlv stages. Established trachoma
progresses through stages I —IV . In fectivity is
m axim um in the early cases. Stage IV is
non infectious.
T h e characteristic
inclusions (Ffelhersfsftfttr flujwawA or H P bodies)
maybe demonstrated in conjunctiva] scrapings, after
Staining hy Giemsa, Castaneda or M a ch i avcl 10
methods.. Because they piHscss a glycogen matlH
they may he stained with iodine which enhances
the sensitivity of smear diagnosis.
'I'he chlamydia may be grown in rhe yolk sac o f
b- ft days old eggs. Thu material is treated with
streptomycin or polymyxin B before inm'LLlation.
T h e eggs are incubated at M ZC in a humid
atmosphere. Blind passages may be necessary fur
isolation. This method is seldom used now as it is
time consuming, cumbersome and relatively
insensitive.
1 isSUC culture using Stationary phase cells
(nonreplicating cells) is rhe method o f choice for
lsol anon. McCoy cells rendered nonreplicating by
irradiation nr antirnetabolitcs arc used. Ile L a or
H L cells treated with D E A E dextran may also he
used. The inoculum has ro be driven into the cells
by centrifugation upto I5,U 00 g to get a good
growth.
Local application and oral
administration o f erythromycin and tetracycline or
other suitable antibiotics should be continued for
several weeV- A singjc-dosc azrtiomycin treatment
has been used with good results.
C o p y rig h te d m aterial
Hidden page

This sexually transmitted disgust, chm illfrisfd by
suppurative inguinal n d c has been kmTwn in
the tropics for a long lime under various names:
lymphogranuloma ingu inaleh poradenitis, climatic
or tropical bubo, It is caused by the L G V Henovtir;
o f Chfoin, ir&chomaii', L l . 1.2 and L3— most
commonly L2. LCVserovanare more invasive than
the other immunotypes o f Chk tilt. trarhaJlUtis.
Their preferred site of multiplication is die regional
lvmph nodes, in contrast 10 T R IC seroviis which
grow in epithelial cells,
I’ he p rim ary legion is a small painless
papulovesicular Icricm appearing on tKc external
genitalia (or rarely cxtragcniral sires) after an
incubation period of three days TO five weeks. The
secondary developing about two weeks later
results from lymphatic spread to ihc draining
lymph nodes. In men the inguinal lymph nudes .Lfe
involved most often and in women the imrapclvic
and pararectal nodes. Women and lioniusejiurd 11Lei i
may develop hemorrhagic proctitis with regional
lymphadenitis, T h e nodes enlarge, suppurate,
become adherent to the skin and break down to
form sinuses discharging pun. M etastatic
com plications may sometimes occur, w ith
involvement o f joints, eyes* and mcningeSr The
tertiary' stage in chronic, lasting for s e ria l vcars,
representing tin: sequelae of scarring and lymphatic
blockage. Late sequelae are more distressing in
women leading to rectal strictures and elephantiasis
of tile vulva (esthiotmene),
T h e prim ary lesion
usually goes unnoticed and the disease is seen
commonly first in the stage o f inguinal adenitis
(bubo), Smears o f material aspirated from the bubos
may show the elementary bodies [Miyagaw.i's
granulocorpusclcs). The sensitivity of microscopic
diagnosis is very low. Isolation of the chlamydia by
intracerebral inoculation into mice and into yolk
sac of eggs has been replaced by cell cultures. L G V
patients develop high title--, of circulating antibodies,
with titles o f 1 ^ 4 or more in C.'F test and Ii5 l2 or
more in m ic ro -IF . Serological diagnosis lS
therefore feasible
A n intradeffllal lest orij^iililllv- described, by FrCb
in 1^25 was commonly used formerly. The crude
chlamydial antigen originally obtained From die
bubo pLic, and later from mouse brnin or yolk sac
cultures (Lygranumnh inoculated intradermally
ill the forearm, with a control on the other aim.
Induration o f 7 m m or more in 2^5 day? was
considered positive. l^uc to the frequent oci'urrence
of false positive reactions, Frci’s test is now not in
imv Treatment is with tetracycline, which should
he given for ar least three weeks.
Psittacosis is a disease of parrots {pSJITftCm means
parrot} and other pstiiacinc birds, transmissible to
human beings. A sim ilar disease acquired hy
nonpsittacinr birds was called ornithosis (omfrAos
meaning birds) but the distinction is now no longer
employed, both condilious being called psittacosis.
Infection in birds is usually subclirtical leading
to a carrier state. Overt disuse may be precipitired
by caging or overcrowing and is manifested as
diarrhea, muLopurluent respiratory disci urge and
emaciation. Chlamydia arc shed in the droppings
ot nasal discharge and aerosols are liberated.
Homan i r tendons are mostly occupational, as in
poultry workers, pigeon farmers, petshop owners,
bird fanciers and veterinarians. I olectioo is by
inhalation. Rare cases o f infection by parrot bites
have been reported. Consum ption o f poultry
products docs not lead to infection, ta re to case
transmission in humans is rare but has been
recorded. The high infectivity Lit' psittacosis is
indicated by the frequency of laboratory infections,
Strains from parrots and turkeys are more virulent
than those from other avian sources.
The incubation period is about ten days. Clinical
disease varies from a mild influenza-like syndrome
to a fatal pneumonia. Though pneumonia in rbe
C o p y rig h te d m aterial
 i C N a m y tfu »
usual clin ical mani trnlat ion, psittacosis i-: a
^]>[iiViitu and may lead to meningoencephalitis
endocarditis, pericardi[i-s, arthritis o ra typhoid-like
syndrome.
L a b o r a to r y d iag nosis: The chlamydia can
be isolated from blood during the early mage* ot
the disease and from sputum later on. Infected cells,
including alveolar macrophages from patients, and
mouse br;ilnh yolk sac and cel! cultures show
inclusion bodies (Lev!ntbsU^Coic—L illie or L C l,
hodies). These differ from C. fradiojnaris inclusion
in being more diffuse and irregular, nor stained by
iodine and nor inhibited by sulphadiazinc or
cycloserine. Tt is generally difficult to recover the
chlamydia from patients titrated wirh antibioses.
Isolation should he attempted only in Laboratories
where special containment facilities are available,
as laboratory infection is a serious hazard.
Serological dli.Lgnos'- may be made by the group
specific C K test or type spc. ific micro-IF.
CHLAMYDIA PNEUMONIAE
C rays ton and colleagues [1 9 8 6 ) isolated a
cLdamydial strain from acute respiratory disease in
adults :.n r.Liwan and designated it as C. psitt&ri
strain T W A R (from Taiwan Acute Respiratory). Jt
possessed the group-specific antigen in common
with C. psj'ftad and C rrachom-ati'. but could be
distinguished from both o f them by species-specific
antigens, D N A hybridisation and resrriction
endonuclease analysis. [ his appears to be an
exclusively human chlamydia transmitted from
human to human without any avian or animal host-
It grows poorly in cell cultures. Because o f these
propertied it has been classified as a separate Species
F u r t h e r It en d in g
Bentty W L et r E, 1 1JI|I I -V r--1■-1^■11: (. .:I.i i i ,-, ,I-..il .I ,l.,i B cv SH tiHti
kiupfiarttft M and P Saikkiu 1V95. Pfwuman.-.i due (d Chlamydia pneumoniae. Clin Infect Di? 21 "Suppl. J, S-2JJ-
I ':-l-li ii:j RW -and RC Bmnham l'iWn. Chbmydiae a pathogen*. Emerging In&it Dif 2:4,
VWiitisrnck 11 cr aJ. 1994. C. erachum, ris infections. Infect Dis Clin ,V ,4m 8;797,
429
called C. pneumoniae.
It appears ro he a common cause o f respiratory
d^casc in older children and adults worldwide.
Antibodies have been demonstrated in the sera o f
about 50 percent o f adults from different parts of
the w orld. Its clinical spectrum includes
pharyngitis, sinusitis, bronchitis and pneumonia,
which resembles jVm»pfa?rna pneumonia. It has
also heen associated with adult onset asthma. 1he
incubation period is 1-3 weeks. OutbreaLs have
been reported in closed communities. Primary
infections occur in young children. Rciilfeclions
ate common- Scrum antibodies do not appear to
be protective.
I )i-ignosis i- hy an| i gen detecti i>n I iv F.I A , ^Ii rect
inimunofluorcscencc or molecular methods, as
isolation o f the organ-‘■m is very d iffic u lt.
Serodiagnosis is by CF, E L IS A or m icro-IF.
Treatm en t La by cute o f [he new m acrolide
antibiotics like clarithromycin or a/ithromvi'in.
Considerable interest has been aroused by recent
reports 11nk: ng C. pnruFminjar with itherusderOM s
and its clinical effects like coronary, carotid and
cerebral arterial disease. Apart from Rtroepidemio-
logical evidence, the finding o f the chlamydial
anil gens in, the isolation nl the organism [rum
coronary artery atheromatous plaques, and the
experimental i iiduciion o f atheroma in rabbrs
infected with the chlam ydia stren g th en the
association, Early results o f a n ti-ch lam ydial
intervention studies in experimental animals and
human volunteers also arc consistent w ith a
causative role o f c . pneum oni.it? in vascular
atheromatous d'sease, However, more work will
have to be done before the issue is finally settled.
C o p y rig h te d m aterial
 General Properties of Viruses
U n ic e llu la r m ic ra o rg u n isms- m ay he classified in
descending order of com plexity as etfJknyorejj, such
as protozoa and fu n g i, and p ro k a iyo res, such as
bacteria, mwtiplasma.-;, rickettsiae and d lb m y d ilC ,
Viruses do n o t fall s tric tly in to rhe category o f
unicellular m icroorganism s as th ev do Tint possess
a c e llu la r o rg a n is a tio n . E v e n rite s im p le s t of
microorganisms are cells enclosed w ith in a cell w all,
c ontaining both types of nucleic acid ( O N A and
R N A ), synthesising their own macromolecular
constituents and multiplying by binary fission.
Viruses, on the other hand, do not have a cellular
organisation and contain only one type o f nucleic
acid, either D N A or R N A lm l never both. Thev
are obligate intracellular parasites- They lack the
enzymes necessary for protein and nucleic acid
synthesis and are dependent for replication on die
synthetic machinery o f host cells. They multiply
by a complex process and no lby biiLary fission.TJ lev
are unaffected by antibacterial antibiotics. I l i e major
differences between viruses and microorganisms are
shown in Table -18.1. In spiLe o f these basic
differences, viruses are generally cons-idered
m krw rpuiinm s in medical microbiology,
Bacteria 4 4 * +
Mycoplasma* t 4 - # +
Riclsettti ae 4 4 * ♦
Chlamydia? 4 - * 4
Viruses - - - -
Viruses occupy the twilight zone that separates
the "living’ from die nonliving’. The demonstration
by Stanley (1935) that viruses could he crystallised
like chemicals, and the extraction by Gcirer and
Schramm (1956) of 'infectious nucleic add' from a
vims that could infect host cells and yield complete
virus progeny made it appear that viruses were only
'living chemicals'. Recent advances in molecular
biology seem to make rhe distinction between 'life1
and W n life ’ little more than a semantic exercise.
As the smallest "living units', viruses offer rhe best
models for understanding the chemistry o f "life".
Ih e medical importance o f viruses lies in their
ability to cause a very large number o f human diseases.
Viral diseases range bom minor ailments such as the
common cold to terrifying diseases such as mbits or
AIDS. They may be sporadic (ike mumps, erwfetTiic
Like infectious hepatitis, epidemic 1he dengue fever or
pandemic like influenza. They may be localised to
circumscribed areas fas some arbovirus diseases) or
worldwide (as I Ierpes simplex). T I k control o f haeK-
rial infection with antibiotics has enhanced the role
o f viral infections in human disease. Viruses can cause
cancer in animals and birds, as well as in humans.
* 4
_
* 4 -
+ -
* 4 4
- - 4
C o p y rig h te d m aterial
 « General Pro p g fllw Of Viruses >
M oRFHO LOGt
Si^-e: The extracellular infection* virus particle is
called [he virion. Viruses are much smaller than
bacteria. It was their small size .mil Tilterabiliry1
(ability to pass through filters that can hold hack
bacter:.:i) that led to their recognition as a separate
class o f infectious agents. Hence they were for a
fin e known as 'filterable vii-uses’. As they were too
small to be seen under the light muroscupe, they
were called 'ultramicroscnpic'. Some o f the larger
viruses, such as poxviruses, can be seen under the
light microscope when suitably stained. The virus
p a rtic lei: seen in th is m anner are know n as
'elementary bodies',
Viruses vjrv widely in sjac. The largest among
them (forexample poxy- ruses) measuring about 300
n il), are as large as the smallest bacteria
(mycoplasma). The smallest viruses {for example
parvovirus) measuring about 20 nm are nearly as
small as the largest protein molecules such as
hemocyanin.
The earliest method o f estimating the size of
virus particles was by passing them through
collodion membrane filters o f graded porosity
(gradocol membranes). The average putt diameter
o f the finest filler that permitted passage o f the
virion gave an estimate o f its size- W ith the
development o f the ultraccntr:fiige, a second method
became available. From the rate o f sedimentation
of virus 1it the ultracentT ituge, the parti vie si ze could
be calculated using Stokes' law, I ’hc third and the
most direct method o f measuring virus size is
cleccron microscopy. Purified preparations of virions
may be examined under the electron microscope
either unstained or stained. By thli method, both
the shape and size o f virions can be studied.
S t r u c tu r e a n d sh a p e : T h e virion consists
essentially «fia nuclei-; acid surrounded by a protei n
coat, the capsid , The capsid with the enclosed
nucleic acid :■■■ known Is the inioftoiwj'fiii. I be
function o f the capsid is to protect the nucleic acid
from inactivation by nucleases and other deleterious
431
agents in die environment. The capsid ii composed
o f a large number o f capsomers which form its
morphological units, The chemical units o f the
capsid arc polypeptide molecules which arc
anranged symmetrically to form an irnpenctrahle
shell around the nucleic acid core (Fig. 4S.1). One
o f the major functions o f the capsid is to introduce
viral genome into host cells by adsorbing readily
to cell surfaces,
1'.vi] kinds o f symmetry arc encountered in the
capsid, icosahedral (cubical) and helical. A n
:C0 ' nhedron is a polygon with 12 verrices or corners
and 20 facets or sides. Each facet iis in the shape ■■I
an equilateral triangle. Two types o f capsnmers
constitute the icosahedral capsid. They are the
pentagonal capsomcrs at the vertices (pentons) and
the hexagonal capsomers making up the facets
(hexons). There are alwavs 12 pentons hut the
number o f hexons varies with the virus group, in
the nucleocapsids w ith helical symmetry, the
capsomcrs and nucleic acid are wound together to
iorm a helical or spiral tube. The tube may be rigid,
as in the tobacco mosaic virus but in die case of
uniiiud viruses, the tubular nucleocapsid ls pliable
and may be coiled on itself. N ot all viruses show
die typical icosahedral Or helical symmetry. Some,
'ike the poxviruses, exhibit a complex symmetry.
Virions may be enveloped or noncnvelopcd
(naked). The envelope or outer covering o f viruses
is derived from the host cell membrane when the
progeny virus is released by budding. The envelope
is lipoprotein in nature. The lipid is largely o f host
cell origin while rhe protein is virus coded. Protein
subunics may be seen as projecting spikes cm the
surface of the envelope. These structures are called
per Jontert (from f ep l usr mean 11 ig envelope). A virus
may have more than one type o f pcplomer. The
influenza virus carries two kinds o f peplomers -
the hemagglutinin which is a triangular spike and
the neuraminidase which is a mushroom shaped
structure. Envelopes! Confer chemical, uiitigenic and
biological properties on viruses. Enveloped viruses
are susceptible to the action a f lipid solvents like
C o p y rig h te d m aterial
 irhNhHAJ.l’KAJPfcKTtl-JiOF Vlkt S.E'.S

P£PLOMER -
B

ENVELOPE
■— CAPSQMERE -
4 NUCLEICACID

— — CAPSID -
A NAKED ICOSAHEDRON

ENVELOPE
\ - CAPSOWERE -
CORE
> - CAPSID —
C. NAKED RIGID HEli X

A - naked 'U 'llnii vim s, cm ixiRljnii nl ;m in iu r t i n iif nuukiL iilm L tiaJiM pd h_v j capsid, mini.' u f
I !■ JillL r-, frurlft A in |-»i■■-■-l- 1n u ,ui C iu d O |K .
C naked hi" I u i; t i n i i ui-iinpji.i.rd nf L-J^um un r■>u i■I i Nl i i .il Ii/ il j l i J H> |i>ihi j lUhnijjl
r> - i7iiiLln|v- lu,'li'.:il virus in 'which lIil Liihul^r LjptiLd i\ plmhlL and i ciK/kiM/d w ilhbi .ir
8,1* ttteP!ii; ji:i'_ S i feo-ifi

ether, ehloroform and bile snlrs.. Specific
neutraljiaricm o f virus infectivity depends on
antibodies to the surface incigtns. UiologiciE
properties sueb a* attachment to hunt ceUsurface
<>r limn agglutination depend an the envelope, borne
viruses possess additional structural features. For
exunple, fibrils protrude from the vertices o f
adenovirus particles.
The overall shiayre o f the virus particle varies in
different groups of viruses, Most animal vintfcsarc
roughly spherical. Some are irregular and
pleomorphic 1 be rubles virus is bullet shaped,
Fbolvirus filamentous and. poxviruses '.■■
shaped' the tobacco mosaic vir i ■ - h ip<
LSacterial viruses have a t imph morphology i |-'i^
48.2).
■ Viruses contain only
one type o f nucleic acid, eubet single or double
‘■ traild v d D N A o r R b J A , En th is re s p e c t, v iru s e s are
unique, for nowhere else in nature i- genetic
i n f o r m a t io n S o lely e a r n e d bv R N .A V i r a l n u c le ic
a c id s m a v b e e x tr a c te d bv t r e a t m e n t v i l l i d e te rm e n ts
or phenol and, in the tafle t -.u- sir iscs :
example picornavims, p. y M . .th e extracted

C o p y rig h te d m aterial
 * General F ir,pefliE5 d VdPUBe6 S" 33

Hg. 41.2 G e ra n iu m sizes and shapes o l ...fa re n l rra tip s ; i1 v ir u :n j. f z iv ln n n 2. R h iiiO iip a
1 H arpasirtfui 4. Aalrovirus 5. T o g n lru i 6. Adanavfaui 7, Parvovirus 9. Pfcom s virus §r BaetartophSQe
10. C o ra ra -ru t 11 Oiihflmyn&vlni; 12- Piravnysionirls'us^

C o p y rig h te d m ate rial

434 ■* Tex'btxjn oF M icr-atiio:-ogy *
nucleic .1. ill lh capableni initiating infection when
introduced into host cells,
Viruses, also contain protein which makes up
the capsid, Viral protein, besides protecting the
nucleic acid, also determines the antigenic specificity
o f the virus. Enveloped viruses contain lipk Is derived
from the host ec]l membrane. Some viruses also
contjiiL small amounts o f carbohydrate. Most viruses
do not possess any enzymes for the synthesis of
viral components or for energy production but some
have other enzymes, for example, the ncuramir.'idase
in the influenza virus. Retrovirues have a unique
enzyme, R N A -dependent'D N A polymerase or
'transcriptase' which can transcribe R N A into
DNA.
R c tis U flC C i W ith Jew exccpl ions, viruses are
very Heat labile. There are individual variations but
in general, they arc inactivated within seconds at
56°C , minutes at 37 °C and days at 4 cC.T1tey are
Stable at low temperatures. For lyri^ term Storage,
they arc kept frozen at -7 0 ''C. A better method for
prolonged storage is lynphilisat'.on or freeze dtying
(drying the frozen vims under vacuum). Lyopliilised
Vi [US can be stored for ye ms and reconstituted when
required by adding water. S i'me viruses (such as
poliovirus] do nor stand freeze drying. Viruses vary
greatly in their resistance to aridity. For example,
enteroviruses are very resistant to acid p H while
rhinoviruscs are w ry susceptible. A il viruses are
disrupted under alkaline conditions.
VLruses are inactivated by sunlight, U V rays and
ionising racial ions. They are, in general, mote
resistant than bacteria to chemical disinfectants,
probably because they lack enzymes. Phenolic
di-i nfcctants are only weakly virucidal. Bacteria are
killed in SO per cent glyceroE saline but this acts as
a preservative for many viruses {for example varan: :S,
rabies). M o la r concentrations o f certain salts
(M g C lj, Na^SQJ also protect home viruses (for
example poliovirus) agninst heat inactivation. The
most active antiviral disinfectants ate oxidising
agents such as hydrogen peroxide> potassium
permanganate and hypochlorites. Organic iodine
compounds arc actively virucidal. Chlorination of
drinking water kills most viruses but its efficacy is
greatly intluenccd by the presence oforganic matter
Some viruses (such as hepatitis virus, polioviruses]
are relatively resistant to ch lorination.
Formaldehyde and beta propiolactonc are actively
virucidal and are commonly employed for the
preparation o f killed viral vaccines.
The action o f lip:d Solvents such as ether,
chloroform and bile salts is selective, the enveloped
viruses being sensitive and the naked viruses
resistant to them. The selective action is useful in
the identification and classification o f viruses.
Aittibioti CS aci. ivc , ilt,iinst bacte ri a are completely
ineffective against viruses. This property i-i made
use o f in e lim in atin g bacteria from clinical
specimens by antibiotic treatment before vims
isolation.
V ir a l H e m a g g l u t in a t io n
V ital lleitiagglulination was originally observed
with the influenza virus by Hirst (1941). A large
num ber of viruses have since been shown to
agglutinate erythrocytes from different species.
Hemagglutination by the influenza virus is due to
the presence of hemagglutinin spikes on the surface
o f the virus. The influenza virus also carries on its
surface another peplom er, the enzym e
neuraminidase which acts on the receptor and
destroys it. Neuraminidase is, therefore, called the
'receptor destroying enzyme' (R D E ), R D E is
produced by many microbes including cholera
vibrios, and is also present in many vertebrate cells.
Destruction o f the receptor leads to the reversal of
hemagglutination and the release o f the virus from
the ied cell surface. This i* known as e/urion.
Hemagglutination if as i convenient method of
detection and assay of the influenza virus. When
red cells arc added to serial dilutions o f a vinal
Suspension, the highest dilution that produces
hemaggtui mat inn provides the hemagglutination
litre. The hemagglutination test can be carried out
in test tubes or special plastic trays. Red cells which
C o p y rig h te d m aterial
Hidden page
 ■" c<tt
■U i t • IDm I k a
- ,.1 ' ‘7"
t( chick nil m
, ' .. ' . i ;' L.J ijli'j I * tt:L
merely of the vital nucleic acid and capsid protein
hut jlso o f enzymes mccesaaiy in die various stages
ofyiral synthesis, asscmbly^d release. In addhiotfi,
certain ' regulator L]is' are also synth raised
which serve to shut down the normal celtubr
m etab o lism and d irect the sequential prod llcLion
o f viral components. The site o f viral synthesis
liepemis cm the type o f virus. In gener.il, must D N A
viruses svnlliesisr their nucleic acid ill the hoit Cell
nucleus. The exceptions are l he poxviruses, which
synthesise all their com ponents in the host cell
cytapbam. Most KIVA viruses synthesise all their
components in [he cytoplasm. Exceptions ire
orthomyxovirustn ami Home paramyxoviruses and
Wims
Influenza virus
E ^ ra in flu e n z a . m u m p s , N 1) V
Measles
Togavirua— ipveml groups nf Arbovirus
Rubella
Enrcntvmis. some Ctwaacltie and E C H O
Rhinovm u, some serotvyes
R a b ie s
Reovirus
M i \*«U*L :b _!iL ’:q dMhwc and iJ% Mpcnstaft
•wv fa dMBaawiMpmt mm prttar onfa Mhm «4dm
mmm Is «flte . =m 1: s 'm m R kgi -j Mtvfifc
retroviruses which nrc synthesised partly in the
nucleus. Viral protein is synthesised only in the
cytP plasm.
Biosynthesis consists essentially o f the following;
steps:
1.Transcription o f messenger R N A (jtlR N A ) from
the vital nucleic acid,
2 .Translation o f die m R N A into 'early proteirak
IThese 'early of nonstnictvraj proteins;' are
enzymes which initiate and maintain Hvntiiesis
o f virus component?. They may also induce
shutdown of' host protein and nucleic acid
synthesis.
.1 Replication a t viral nucleic acid-
hbwli, human, guinea pig, otheri; Elution at 37 "C
fow l human. guinea pii', others; Llution at 37 "C;
Htntuly^iti present
Monkey, 37 "C
Goose, pigeon, one kl ,iy old chick; pH and
[empennnue critical
Goose, |>jgcunFtmetUy old chick; 4*C
I lum.in; 4 "C and .37 "C
sheep; 4 '‘C
Goose; 4 ->C, pH t,2
I luman; .37 ‘K?
C o p y rig h te d m aterial
4. Synrhesis o f LLater or structural proteins, which
lire the components o f daughter virion capsids.
T h e critical step in viral biosynthesis is the
ETairs erip tiijfi o f CnRNA from the viral nucleic LLrid.
O eicc tills: ]■-. achieved, the hunt ceil ItBourceS can
he utilised for tran slatin g m R N A into viral
components. Depending cm the structure o f then
genom e, viruses use dlllercnt strategies tor the
tra n scrip tio n o f m R N A . V iruses have been
categorised into six classes by Baltimore (1'>7U'}
ha^ed on rhier replication mechanisms.
In rhc cp sc o f fully double stranded
DNA viruses (su ch as a d e n o -h herpes-^
papovavi ruses), the D N A enters the host cell
nucleus and uses th e host cell enzym es tor
transcription.The extracted DNA from these viruses
is infectious. W ith hepadn Liviruses which have a
partially double stranded D N A , the duplex is
completed by a viral D N A polymerase, ]uside the
host cytoplasm. "The mature DNA then moves into
the nucleus, to be transcribed by host transcriptases,
Extracrcd hepadnavirus D N A is not infectious.
Poxviruses which replicate In rhe cytoplasm form
m R N A using polymerases contained in rhe virion
itself l\jxvirus DNA is nut infectious.
W ith singlt' stranded DNA viruses (for
example parvovirus), the D NA molecule moves into
the host cell nucleus and is converted into the duplex
form. Transcription is achieved by host enzymes.
In reoviruses, the double Stranded RNA
is transcribed to- m RN A by viral polymerases.
Depending on the m ethod o f m R N A
transcription, single stranded R N A viruses arc
classified, into two categories. In the posiOit sXra.nd
(plus fitmntl, pO$iQw sense) RN A viruses, tile; viral
R N A itself act as the m R N A . Vririri R N A is
infectious bv itself and is translated directly into
viral proteins in rhe host cell cytoplasm (for example
picorna-, Eogaviruses).
Tlte itegaj;ve siramJ (minus sense) RN A
viruses (fo r exam ple r bah d o -, o rfh o m y x o -,
param yioviridac} the R N A is ‘antisense', with
polarity opposite to mRNA^Thcy possess their own
R N A polym erases for m R N A tra n scrip tio n .
Extracted nucleic acids from these viruses arc not
infectious.
RetroviridiLe exhibit a unique replicative
strategy. T h eir single stranded RN A genom e is
converted into an R X A lDNA hvbrid bv the viral
reverse ttanscfipt& se (R N A d irected D N A
polymerase) enzyme. Double stranded DNA is then
synthesised from the R N A lD N A hybrid. The
double stranded D NA form o f the incus (provinis)
is integrated into the host cell chromosome. This
integration may Lead to transformation o f the cell
and development o f neoplasia.
A ssem bly o f d au g h ter virions
follows the synthesis o f viral nucleic acid and
proteins. Virion assembly may rake place in the
host te ll nucleus o r cytoplasm - H erp es and
adenoviruses arc assembled in the nucleus, while
p icorn a and poxviruses are assembled in the
cytoplasm- At this stage, the nnnqnvelnpcd viruses
are present intracellularly as fully developed virions
but in the case o f enveloped viruses, only the
nucleocapsid is complete. Envelopes are derived
from the host cell membrane during die process o f
budding. The host cell membrane which becomes
the envelope is modified by incorporation of vims-
spccifk antigens. Herpes virtis« assembled in rhe
nucleus acquire their envelope from the nuclear
membrane as they are released into the cytoplasm
enclosed in a vesicle, Myxovimscs bud from the
cell surface and their envelope is formed by the
modified cytoplasmic membrane o f the host cell.
The incorporation o f viral antigen (hemagglutinin)
on the cell membrane endows the cell with the
property of hemadsorption.
In the case o f bacterial viruses, the
release of progeny virions takes place by the lysis
o f the infected liacBerium. However, in the case of
animal viruses, release usually occurs without cell
lysis, Myxoviruses are released by a process o f
butlding from the cel] membrane over a period of
time. The host cel] is unaffected and may even
divide, the daughter cells continuing to release
Copyrighted material
Hidden page
Hidden page
 system generally constating of bicarbonate in
equilibrium with atmosphere containing :iboe]T
5% carl^m dioxide. This is Supplemented with
UptO 5% ClEf Of fetal calf sen] m. Antibiotics .ure
added to prevent hacterial contaminants aud
phenol ted ns indicator. Suck media wdl Citable
most cell types to multiply with a division time
of 3 4 -4 8 hours, "lire cell suspc nsion Is di apensed
i n bottles, tubes. ur Pttr i dishes. The cells adhere
to the glass surface and on incubation, divide to
form a confluent m onolayer sheet o f cells
covering the surface within ahour a week.
Cell culture tube- may he incubated in a sloped
horizontal position, cither as "stationaiy culture'
or may he rolled in special 'roller drums' to
provide taller aeration. Some fastidious viruses
grow only in such roller cultures,
based on their origin, chromosomal characters
and the mimhcr of generations through which they
can be maintained, cell cultures lire classified into
three types (Table 48,3):
These are normal cells
freshly taken from the body and cultured. They
arc capable of only limited growth in culture
and cannot be maintained in serial culture.
Com m on examples of primary cetl cultures ire
monkey kidney, h Liman em bryonic kidney,
human amnion and chick embryo cell cultures.
Primary cell cultures arc useful for the isolation
o f viruses and their cultivation for vaccine
production.
These are cells of j single
type th At reta in the origi nal diplo id chromosome
num ber and karyotype during serial
Su.bc ulliValioO for u. him Led number of times.
After about fifty serial passages, ihey undergo
'senescence'. Utploid strains developed from
human fibroblasts arc susceptible to i wide range
ot human viruses and arc very useful for the
isolation o f some fastidious pathogens. They .lcc
also cm [i loved for the product ion o f viral
vaccines.
These arc cells nt 2 single
type, usually derived from cancer cells, that are
capable o f Continuous set Lai Cultivation
indefinitely. Standard cell lines derived from
human cancers, such as H rL a , H ep-2 and KFS
cell lines hive been used in lab o rato ries
throughout the world for many years. These cell
lines may be maintained by serial suhcultivation
or stored in the cold ( - 7 0 *C) for use when
necessary. Some cell lines are now permitted to
he used for vaccine manufacture, for example
Vetoed I tor rabies vaccine.
Vims growth in cell cultures Can he
detected by the following methods:
M an y viruses
m orphological changes in cultured cells in
which they grow. These changes c a r be readily
observed by microscopic examination o f the
ciiltures. These charges ire known as cytopalhic
effects' (C P E ) and the viruses causing C P E are
called 'cy to p ath o g en ic viruses’ . T h e C P E
produced by d] fife rent groups o f viruses are
ch aracteristic and help in the presum ptive
identification of vims isolates. For example,
enteroviruses produce rapid C P E with crcnation
of ceils and degeneration o f the entire cell sheets
measles vims produces syncytium formation;
herpes virus causes discrete focal degeneration;
adenovirus produces large granular clumps
resembling bunches o f gripes; and SV^ produces
prominent cytoplasmic vacuolatlon.
In normal cell cultures,
the m edium turns acid due to cellular
metabolism. W hen viruses grow in cell cultures,
cell metabolism is inhibited and there is no acid,
production. This can Ita made out by the colour
o f the indicator (phenol red) incorporated in
the medium.
W hen hcmagglutinating viruses,
(such as influenza and parainfluenza viruses)
grow in cell cultures, their presence can be
in d icated bv the addition o f guinea pig
etytbrocytcs to the cultures. I f the viruses arc
Copyrighted material
 multiplying in the culture,, the erythrocytes will
adsorb onto the surface o f cells. This lh known
as 'hemadsorption'.
The growth of a nran-cytoparbogcrk
virus in cell culture can he tested by the
subsequent challenge w ith i known
uytopathngcnic virus. The growth of the first
will inhibit infection by the second vims by
interference.
Tumour forming (oncogenic)
viruses induce cell "transformation' and Doss of
contact inhibition, so that growth appears in a
piled-up fashion producing 'micTonimours’.
Cells tmm vims infected
cultures can be stained by fluorescent conjugated
an tiseru m and exam in ed under the UV
microscope for the presence o f virus antigen.
This gives positive results earlier than other
merhods and, therefore, finds wide application
in diagnostic virology,
en u m eration are electro n m icro sco p y and
hemagglutination. Ry simple negative staining* the
virus particles in a suspension can be counted directly
under the election microscope.The virus suspension
can be mixed with a known concentration o f latex
panicles The ratio between the virus and latex
particles under the electron microscope gives iin
in d ication of the virus cou n t- W ith
hjemagglutinating viruses, a convenient method of
q u an titatio n is the de term i n atio n of
hemagglutination titres. Hcmagglurination is not
a very sensitive indicator o f the presence o f small
amounts o f virus particles. Thus, approximately 1 U:
influen za virion s are required to produce
macroscopic agglutination of a convenient quantity
of chicken erythrocytes (0 .5 ml o f 0 .5 per cent
suspension}. However, because o f its simplicity,
hem agglutination is a very convenient method of
virus assay;

lwo types o f inirctivity assays can be carried out -

T h e virus content o f a specimen oar be assayed in quantitative and quintal assays. Quantitative assays
two w ap: cither with reference to the total vims measure the actual number o f infectious particles
particles or with reference to the infectious virions in the inoculum, while quanta! assays only indicate
only, Two methods employed for total particle the presence or absence of infectious viruses. Using

a- Primary' cell culture?

1. Rhesus monkey kidney cell culture
2 . Human amnion cell culture
uChick embryo fibroblast cell culture
b-Diploid cell strain*
1. W T 3 8 Human embryonic lung cdl strain
2. HL-3 Khtwus embryo cdl strain
c Contimiouscell linn
1 . HcLa l Ivman carcinoma of r t ™ cell line
2 . H F P -2 Homan epithelioma o f larynx cell line
X KB Human carcinoma of nasopharynx cell Line
4. McCoy Human synovial carcinoma cell line
5. Derroit-6 Sternal marrow cell line
6. Chang C W L/K Human coniunctiva (C)
Intestine (I), Liver ^L) and Kidney (K) cell line*
7. Veto Vcrvel nwribey kidney cell line
Baby hamster kidney cell line

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 GEN h k A l. HKlSPERTIE5 OF V IRUSES
C^OR|OALLi-‘ .'T0:C
MEMBRANE
A1FI 7jAC
AM NIO TC C A 1 r V
S he ll
SHf '. l
MEVBHANE
A, LAMO C
LAVIT V
YO.* SAC-
ALEUMCM
icrk l dilurioi . o f virus suspensions uui with the
aid o f statisi ical niLthods, reasonably accurate
estimates nf inactivity can he obtained "m quanta]
im p .
Qimull] M i p o f infect ivitv can lie curried Out
in animals, eggs o r tissue culture. Exam ples of
endpoints used for infeedvity titration are the death
o f the anim al, production o f hemagglutinin in
allantoic fluid or the appearance o f C P E in cell
cultures. The titre is usually ^pressed as the '50
per cent infectious dose (I D q||) per ml, which
indicates the highest dilution o f the inoculum that
vw ld produce an effect in SOper cent o f animals,
■ggs Of cell cultures inoculated. ID... i$ calculated
ny the application o f statistical methods, such as
that o f Reed and M ucnch.
The quarifPt.itjVe i"jlfochvr"ty assay of viruses is
similar to th e estimation nf bacterial viable counts
by colony counting. Two methods are available -
plaque assay in monolayer cell eulturc m J poclc
assay on chick embryo C A M . Plaque as.yv was
introduced in an irunt vi fotogy bn' DulbdCCO (19 5 2 )
as a ruoilil icatii hi o f die bacteriophage plaque assay.
A vital suspension is added to a m onolayer of
cultured cells in a horde or Petri dish, and after
allowing tim e for absorption, the medium is
removed and replaced with a solid agar gel, to CHUUK
that the spread [if progeny virions is confined to
rhe immediate vicinity o f Lnfiecttd cells. In this
system, each infectious viral particle gives rise ro a
localised focus of Infected cells that can he seen
with rhe naked eye. Such foci arc known as
'plaques’ and each plaque indicates an infectious
virus (b ig . 4 8 .5 } . Som e viruses w hich are
transmitted directly from cell to cell (for example
herpesvirus) may form plaques even without an
agar overlay. O n co g en ic viruses produce cell
transform ation which can be hceh as mictO-
fumnurs. Hence they can be enumerated by the
transformation assay.
Viruses that form. pack:-, on C A M (for example
vaccinia) can be assayed by counting the number
o f pocks formed on C A M by appropriate inocnla
o f virus. This is known as pock assay.
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Liter all other 'lining beings', viruses obey the laws
o f gpncti.es. Several properties o f viruses, such as.
virulence and antigenicity, that arc o f great concern
to human beings in the context of infections at the
level of the cell, individual .md [□mmunity, are under
genetic control. G enetic studies, therefore, have
contributed to a better understanding o f vtnw-hosi
interactions and the development o f better viral
vaccines. Genetic mechanisms such us mutation and
s e le ctio n w ere utilised in the past w ith o u t
recognising [he biological mechanisms involved.
T h e development o f the 'fixed' rabies virus by
Pasteur is a case in point.
T h e two m ain m ech an ism s fur genetic
modification in viruses are mutation and
recombination. In addition, viruses may exhihit
many noTiheritablc variations due to gene product
interactions.
T h e frequency o f mutation in viruses
is about 10 * rn 1 11 approximately the same as in
bacteria. Mutations, therefore, occur during every
viral infection. M ost mutations arc lethal. A mutant
becomes evident only if the mutation confers some
readily observable property or survival advantage.
M utation may occur spontaneously or may be
induced by m utagens, physical agents such as
irrad iatio n o r ch em ical agents such as 5 -
ftunrouracil-
Thc mutants may he o f various types- Some
mutations o f clinical and laboratory interest arc
those affecting virulence, host range, antigenicity
and pock or plaque morphology. A class o f mutants
that are o f great importance in laboratory studies is
the condrfiorrid le t h a l .mutanr. These are mutants
which arc able to grow under certain conditions
(called permissive conditions), but arc lethal, that
is it cannot grow under certain other specified
conditions (called non permissive or restrictive
conditions). There are different types o f conditional
lethal mutants but the types most widely employed
in genetic studies are the 'temperature sensitive7 [ts)
mutants. These can grow at a low (permissive)
temperature (2fl."3I'>1C ), but not i t ;i higher
(restrictive) temperature (3 7 “C ). The advantage
here is lI i .lL by u sin g a single selectiv e test
(temperature sensitivity), laqre numbers of mutants
with lesions in different genes may be obtained.
T h e £f mutants have not only contributed largely
to fundamental studies on viral genetics but also,
because o f their low virulence, offer prospects of
better live viral, vaccines.
G enetic recom bination may
occur when two different, but related, viruses infect
a cell simultaneously. T h e two viruses exchange
segments o f nucleic acid between them that a
hybrid results, possessing genes from both parents.
Such reco m b in an ts breed tru e th ereafter.
Recombinants may occur between ( 1 ) two active
(infectious) viruses; (2 ) one active and one inactive
vims; and (3) two inactive viruses.
W hen two different strains- o f the same yitu4
(such as vaccinia or influenza), possessing distinctive
markers (such as pock morphology or antigenic
properties) ate grown together, recombinantH may
hr derived that possess the distinctive properties of
both parents. Thus, if a human and an avian attain
o f influenza virus (whose hem agglutinin and
neuraminidase antigens arc different and easily
identifiable) arc grown together, a hybrid may be
obtained with the hemagglutinin o f one parent and
the neuraminidase o f the other. This has been
demonstrated experimentally in vitro and in vivo,
This may be one o fth e ways by which the pandemic
strain o f the influenza virus originate in nature.
W hen a cell is ■infected" with an active virus
and a different but related inactivated virus, progeny
possessing one Of m ore geilC tk fruits o f the
in activ ated virus may be p rod u ced . T h is
phenomenon is called entss reactivation or marker
rescue. Mew antigenic variants at the influenzaVIIU5
causing epidemics, often do not grow well in eggs
as com pared to established Laboratory strains.
W hen such an epidemic strain (for example strain
A J is grown in eggs along with a standard strain
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 u 4 ..
■ ’' ! ■!. ; i
Till jliout ] 9S 0 little.1 wap known ol the baton
p ro p erties viru ses. J’hey were nam ed
haphazard based un lUl- diseases i k v caused or
on the place o f their isolation. The}' were grouped
According ro assumed "tropisms1 nr affinity to
different systems o r organs o f the body. Thus human
viruses were classified as dcrmotniplc, that is those
producing skin lesions (sm allpox, dtickcnjH ^,
measles), neurntropic, that is those affecting the
nervous system ( polio m y elitis, rab ies},
pneuniutropk, that is those affecting the respirators
tract (influenm, common cold) and visetrotropic,
that is those affecting visceral organs (yellow tever,
hepatitis). Rawdcn (1 9 4 1 ) made the pioneering
suggestion that viral nomenclature and classification
should be hnijijJ on the properties o f viruses and
not upon host responses. From the early 19150s,
0- ■>
viruses hegail to be dassilied itlTO groups based Otl
their physicochem ical and structural features.
Ni Mnendatunc arid class illcation atc notv the official
responsibility of the International Committee on
Taxonomy o f Vimses.
Viruses .ire classified into two main divisions
depending on die type o f nucleic add they i Assess:
fibpvi rusvs are thusc c o n ta in in g R \ A and
Jeo xy n b o v i ruses arc [hose con tain in g O N A.
Further d as-ilivarion i- based oil other properties sucii
as rhe sfrandedress o f micfeic acid* symmetry o f
nudetKapsidn presence o f envelope, size and shufn: of
virion and number ot cap-timcn. Short desmptioiw
djfthc major groups o f viruses arc given below.
T h e se are large, brick
shaped or ovoid viruses (300 * 240 * UKl p), with
complex structure, hiving a lipid containing an
outrr coat, one nr two Lateral belies and a enrr
carrying a single linear molecule o f double stranded
U N A . Multiplication And maturation rake place in
the cytoplasm. The family is divided into several
genera,
■' ■ T h ese are m edium
%ivcd i-imhe-i containing linear double stranded
UNA. The icosahcdral nuckocapud ilOH nm) has
U?2 capKomers and is surrounded by a. Lipid
antfliiung envelope. Multiplication lakes place in
the nucleus and maturation by building through
th e n u clear m em b ran e. O n ly one gen u s.
H crpesvifm , has been characterised hut several
members o f the tamiiy await classification.
T h ese are m edium
M2 td (7 0 -9 0 nm } nqtaenweloped, kosahedril virtises
with 252 capsotriers. Members have heen classilied
into two genera: M astadcn ovinia (m am m alian
adenoviruses) and Aviadcnovirus (adenoviruses of
birds)
'Hiese arc small ( 4 0 -
55 nm) noucnvelopcd, double stranded D N A
viruses with 12 eapsom m . Iwo gtneni have hcen
rtCogoised, PapiHoftigvjtiia anti fWyorna rirtii.
C o p y rig h te d m aterial
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 Virus-Host Interactions: Viral Infections
V iru s -host interactions m i)1 be considered at
different levels - rbe cell, the individual and the
community.
A t the cellular level, viral infection may cause a
broad spectrum of effects, ranging from no apparent
cellular damage to rapid cell destruction. Some
viruses, like poliovirus cause cell death (cytocidal
effect) or even lysis (cyrolysis). Others may cause
cellular proliferation fas molluscum contagiosum)
or malignant transformation (as oncogenic viruses).
In some instances the virus and hosEuell enter iota
■a peaceful c o e x iste n c e , both rep licatin g
independently w ithout any cellu lar injury, a
condition known a s ‘steadv state infection'. In tissue
culture, viral infection may lead lo readily observable
cellular changes (cytopathic effects). These may not
parallel tlie changes produced in the infected
anim al, as in the latter situation infection lH
influenced by the various defence mechanisms o f
the body.
Cellular injury may be due to a number o f causes.
Early or iwiumirtural viral proteins often cause a shut­
down of host protein and D N A synthesis. Large
amounts of vim! mactofnoleculfi tliut accumulate ill
the infected cell may distort the cellular architecture
and exert a toxic effect, Tlie permeability o f plasma
membranes may be altered, releasing lysosomal
enzymes and leading to autolysis,
M a n y viruses p ro d u ct alte ra tio n s in th e
cytoplasmic membrane o f infected cells. Some (such
as respiratory syncytial virus) cause fusion o f
adjacent cell membranes, leading to pofykaryocytosis
or syncytium formation. Vims coded antigens may
appear on the surface o f infected cells. These
antigens may confer new properties on the cells.
For example, viral hemagglutinin appears on the
surface o f cetla infected with influenza virus and
causes adsorption o f erythrocytes to the Cell surface
(hemadsorption}. Virus coded antigens also appear
on the surface o f cells transformed by oncogenic
viruses.
C e rta in viruses such as m easles, m um ps,
adenoviruses, cytomegalovirus and varicella virus
cause damage to the chromosomes o f host cells.
Chrom atid gaps and breaks in chrom osom e 17
occur frequently in cultured cells infected with
adenovirus ty^ies 1 2 and ill.
The most characteristic histological feature in
vims infected tell* is the appearance o f WltJusion
bodies. Inclusion bodies arc structures with distinct
size, shape, location and staining properties that
can be- demonstrated in vims infected Cells under
the light microscope- They m aybe situated in the
cytoplasm (as w ith p o xv iru ses), nucleus
(herpesviruses) <>r both (measles virus). They are
generally acidophilic and can be seen as pink
structures when stained with G iem sa or cos in
methylene blue stains. Some V] ruses (for example
adenovirus) form basop h ilic in clu sion s.
Demonstration o f inclusion hodies helps in the
diagnosis o f some viral infections. The presence at
intracytoplasmic eosinophilic inclusions (N egri
bodies) in the hrain cells o f animal? justifies the
presumptive diagnosis o f rabies. Vaccinia infected
cells show rather smaller multiple inclusions known
as Guarrieri bodies. Large inclusions (Bollinger
bodies) arc seen in fowlpox. Inclusion bodies in
molluscum contagLoaum (nnolluscum bodies) are
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45G < Teurcook d MicrDtWolDgy ►
very Large (20- JGjJm) andean he itadilySecn under
tile low power microscope, imranuclcai : - iclusio-n
bodies ■were clas-iiied into two types by Cowdrv
(1^.14). Cowdry type A inclusions are o f variable
sl7,c and granular appearance (as with herpesvirus,
yellow fever vims), while type R indusions are more
circu m scrib ed and o ften m ultiple (as w ith
adenovirus, poliovirus), Inclusion bodies may be
crystalline aggregates o ft i rions or made up ofvims
antii^ens present at the Mte o f virus synthesis. Some
inelunions represent tlegenerativc changes produced
by viral infection winch confer altered staining
properties on the cell.
P a T H O G F N F S IS OF \ IR A L IN F E C T IO N
Depending on the clinical outcome, viral infections
can be cl as--1 feed as inapparrnt (subclinical) or
apparent Iclinical or overt) infection*. The latter
may be acute, subacute or chronic. Some virus
infections are characterised by latency. L aten t
infections are o f different types. Recurrent herpes
simplex, and l:u pc- zoster are examples of Latent
infections in wiii.h clinical manifestations appear
after prolonged periods o f quiescence during
which the viruses remain hidden in the nerve
root gangli.i. Another type o f latent infection is
p erh ren t tolerant infection in which the virus is
readily demonstrable in the tissues o f the host but
n a th e r disease nor immune response develop®. The
host is nnmunnlngicaLLy tolerant to the virus as a
result o f congenital or neonatal infer non- Disease
sets in when [be tolerance is interrupted. The
cLassi'id example o f persistent tolerant injection is
lymphocytic choriomeningitis of mice. Another
type o f latent infection is seen in neurological
diseases such as scrapie in sheep and Icum
human beings. T*his is called slowly progressiv e or
slow infection as the incubation period is unusually
long. Vet another class o f latent infection is infection
by on cog en ic viru ses. The hum an
immunodeficiency vims leads to a special type of
latency, with an initial asym ptom atic period
followed by progressive immune damage causing
secondary diseases, ending fatally after many
years.
Viruses enter the body through the respiratory
and aiimentarv tracts, skin, conjunctiva and the
genital tract. Many viruses are transmitted vertically
from parent to pmgeny.
TTic respiratory tract offers the most important
portal of entry for viruses. A Large nmnlier o f viruses
arc able to infect the cells o f th :s tract. Some of
them multiply locally Lo initiate a silent local
infect inn w hich is follow ed by Lymphatic nr
hematogenous transport to other situations where
m ote extensive multiplication takes place before
system ic illness is m anifested. Sm allpox and
chiekenpox. arc examples o f such systemic diseases
in which the portal o f entry is the respiratory tract.
O ther viruses such as influenza and rh i novi ruses
are restricted tn the respiratory tract where they
m uliiply and produce local disease. These are
known as respiratory vbuses,
The alimentary tract is the next most important
mute o f entry for viruses. However, only some
viruses are able to establish infection in the
intestines. All enveloped viruses are destroyed by
bile. Rhinoviruses are inactivated by gastric
acidity. Only enteroviruses, adrnovi ruses, rrnviruses,
h ep atitis viruses and the viruses cau sin g
gastroenteritis are able ro set up intestinal infection.
Some o f these such as rotavirus nemaio confined
tn the gut causing local disease. O thers such as
poliovirus) after i"iir-al multiplication locally, art
transported to other sites for further multiplication
and subsequent spread tn the target organ 5.
O f the viruses that enter through rhe skin,
only a few produce Local Lesions. Papillom a,
in vaccinia, cowpox, naolluscum contagiosum and
orf are viruses that produce dermal legion-: at
the site oi entry. Skin lesions o f exanthematous
viral diseases arc secondary ro systemic infection.
V iruses en ter the skin th rou gh abrasions
(papillomavirus), insect bites (arboviruses), animal
bites (rabies) or injections (type B hepatitis),
Systemic spread occurs through lym phatics or
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blood. Rabies virus travels along the nerves to the
spinal cord or brain.
T h e am j umetivj also may act as a portal o f COEry
fur viruses. T h is m ay lead to local disease
(adenovirus) or lo systemic spread (measles). Some
viruses may enter through the genital tract or other
sites o f sexual contact (H IV ).
Congenital infection may occur at any stage
from tlir development yf the ovum up to birth, lo
acute system ic infections, congenital infection
usually leads to fetal death and abortion. Rubella
anti cytomegalovirus produce iTLBJdcvelopment or
severe neonatal disease. Vertical transmission is the
natural mode o f spread o f many tumour viruses.
The avian leulufsis virus in transmitted in ovo and
murine m ammary tumour virus through breast
milk.
Fhc manner in ivbich the infecting VtTufi S]7ieads
from the poi nt o f e ntry, multipl ics in sires o f election
and causes lesions in target tissues was first studied
by Fen n er ( 1 9 4 8 ) using m ousepox as the
experimental model (F ig , 4 9 .1 ). T h e muu-ScpOJf
vims enter? the --kin, where it multiplies initially
and proceeds along the lymphatics to the local
nodes. After multiplication in tile lymph nodes, the
virus enters the bloodstream (primary viremia) and
is transported to the spleen and liver which act an
the ‘central foci' for viral m ultiplication. A fter
extensive multiplication in the central foci, there
occurs a massive spillover o f the virus into the
bloodstream (secondary viremia). This heralds the
otiier o f clinical symproms (die prodromal phase
in eruptive fevers). The virus reaches the target
organ (skin in eruptive fevers) th rou gh the
bloodstream . M ultiplication in the target sites
produces the distinctive lesions. W ith m inor
m odifications, thin model holds good for m ost
system ic viru s diseases. T h e reasons for the
difference in the foci, o f multiplication and target
organs in the case o f different viruses are
obscure.
The incubation period represents the time taken
for the virus to spread from the sire o f entry to the
organs o f viral multiplication and thence to the
target organs f ir the production o f lesions. Its
duration is therefore influenced by the relation
between the sites o f entry, multiplication and lesion.
W here the site o f entry and site of leeiotl arc the
same, tlie incubation period is short— one to three
days, as in respiratory viral infections and in
gastnrenteritin. In systemic diseases where lIll: Virus
enters through the respiratoty or alimentary tract
and produces lesions in rem ote target sites, the
incuh&tion period is Io n g re-^ 10 —2 0 days, as in
chickcnpox or poliomyelitis. There are, however,
exceptions to this rule. In arbovirus diseases, as in
yellow fever or dengue* the incubation period may
he shorter If fi days), probably because the virus
is introduced directly into the bloodstream by the
insect vectors, T h e incubation period in type R
hepatitis may be 2 -f> m onths and in slow viral
infections, many years. Papillomas and molluscum
contagiusum have long incubation periods, probably
because the viruses multipty slowly.
The outcome o f a viniR Infection is influenced by
the virulence o f the infecting strain and the
resistance offered by the host. Mechanisms of host
resistance may be immunological or nonspecific,
T h e la tte r includes various g e n e tic and
physiological factors such as interferon production,
body temperature, nutrition and hormones.
V irions in
genera] are good antigens and induce both humoral
and cellular immune responses. The multiplication
o f a yin1- in tlie body during infection induces iot
only a in ..mill lively greater imn nine response out
also liberates and makes available to the immune
system the whole range o f vims antigens, including
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 TO riOOK t - V Mlt'KOHIULOGV

SweSog Crf fitftllTl

I’ooT-P-rm^v leMai

Early rSSlv
papties

Fig. 491 PAtfMfttrtuli a1 fflaLtMptaH—a mcrtM for x u l* totfrtlhMUta tf luuttirt* (tfUr Fcnnttr)

su rface and iurcrna] nnri^ci’.s as wql] as the
nonstructutal antigens such as early proteins.
In mediating humoral antiviral immunity; the
important classes o f antibodies arc IgC , JgM and
IgA. IgG and Ij^M play a major role in blood and
tissue spaces, while IgA is mure im portant on
m u cosal su rfaces. A n tib o d ies el feet vim s
neutralisation bv
f several mechanisms. Thev
T
may
I
prevent adsorption of rhe virus ri> cell receptors,
cause enhanced virus degradation ot prevenr release
ot rhe progen y virus from in fected cells.
Complement acre in conjunction wirlt antibodies
in causing surface damage lo enveloped virions and
in producing cvtolysis ol virus infected cells.
Mot all antibodies arc able to neutralise viral
infect! vity, Antibodies to internal antigens are non­
 ■I Virus-Host Interactional Vral Infections *
neutralising. Antibodies to surface antgens vary in
then: neutral i-. mg ability, For instance, two types of
surface antibodies appear follow lrij^ influenza
in fectio n — an tih cm ag g lu tin in and antirLeur-
nminidase-'l^ie termer can neutralist' irfectivity hut
the latter cannot. Antincuraminidasc antibody car,
however, inhib't the release o f progensd virions from
infected cells. Some antibodies can paradoxically
enhance viral mfectivity. Humoral anti Iusd its m±y
sometimes actually contribute To pathogenesis.
An \ib™lies may cause an :i] feme i tie]lendent i:i jury
to cells or induce an immune complex type o f rissue
inj ury. The enlianced severity o f respiratory syncytial
viral infection in early infancy i.s believed to be due
to the presence o f passively acquired m aternal
antibodies. In older children who have no antibody,
the virus causes a milder disease. The pathogenesis
o f som e viral hem orrhagic fevers is immune
thrombocytopenia. M ost extrahepatic lesions in
type R hepatitis are due to damage caused by
immune complexes.
Cell mediated immunity is o f criLical importance
in viral infections-The earliest indication tif cell
mediated immuniiy in viral infection! was the
demonstration of delayed hypersensitivity following
vaccination in immune individuals. Similar skin
reactivity is also seen in mumps,. T h e normal
resistan ce to virus in fectio n s show n by
again m iglobulincm ics is ascribed to their cell
mediated immunity, though M
. may also be due to
interferon o r other non immune m echanism s.
Individuals with deficient cellular immunity show
a heightened susceptibility to infection by the
herpes, pox and me- isles viruse ■■.The udim 11 istra i ion
o f antilymphocytc scrum induces fatal infection in
mice injected with a sublechaldose oftheectrom elia
virus. Cell mediated immunity is considered to piay
a major role in recovety from viral infections in
which vincmi:i is not im portant and in which
infected cells have viral specific antigens on their
surface. In some virus infections cell mediated
immuniiy may contribute to tissue damage, as n
lymphocytic choriomeningitis in mice.
4*3
Some vira) infections cause a suppression o f rhe
■mmune response. Measles infection induces a
temporary depression o f delayed hypersensitivity to
tuberculin. Infection o f adult mice with lymphocytic
choriom eningitis or leukemia viruses inhibits
antibody response to other antigens. H IV strikes at
the centre of the immune ^stem hy infecting [be
CD 4+ helper T cell.
In general, viral infections ire followed by solid
im m unity to reinfect ion, which may often be
lifelong. AppaTC nl exceptions !like the com mon cold
and influenza arc not due to lack ol immunity hut
to reinfection being caused by antigenically different
viruses, Live virus vaccines also induce more durable
protection than bacterial vaccines.
N o n im m u n o lo g ica l R e s p o n s e ?
L Phagocytosis: Polymorphonucleai leucocytes do
not play any significant role in the defence
against viral infections. In feet, more viral
diseases arc ch a ra cte rise d by a
polymorphonuclear leucopema. O n the other
band, macrophage:- phagocytose viruses and are
im p o rta n t in clearin g viruses from th e
bloodstream.
2 . Bodyr temperarurr Fever may act u a natural
defence mechan i-m against viral infections as
most vi ruses are inh ibited by temperatures above
3 9 C . An exception is herpes simplex which is
usually reactivated by fever to produce ‘fever
bl isters\ H erp es f e b r.'.’rS is a freq u en t
accompaniment o f fevers caused by pneumococci „
s tr e p to c o c c i, influenza virus and m il an a
parasires, bur for some unknown reason, is very
rare in other fever? {typhoid, tuberculosis).
3 , W o ra to u e c C o rtico ste ro id adm inistration
enhances most viral infections. Consackie virus
E l does not nurmally cause disease in adult mice
but will induce a fatal infection in mice treated
with cortisone. Normally mild infections such
as varicella and vaccin ia may be lethal in
patients on cortisone. Injudicious use of steroids
in the Treatment o f herpe tit keratoconjunctivills
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 4 Virus-Hgsl Interactions: Viral Intficliana t
epithelial cells following stimulation by viruses or
polynucleotides. It is a glycoprotein.
Gamma inlfcffertw (IF N -7 ), formerly known as
b m itn e interferon, Is produced by T lymphocytes*
oil Stimulation by antigens o r rmtogtnS. It i<i a
g ly co p ro te in . It is m ore co n cern ed witti
i inmunomoduktoiy and antiproliferative functions
than with antiviral defence, It also differs from alpha
and beta interferons in having a separate cell
receptor.
In terfero n s are in activated by prnteolytic
enzymes but not by nucleases of Hipases. Tkev resist
beating at 5 6 —60 5C for 3 0 —60 minutes and are
stable over a wide range o f pH (2 - 1 0 ), except
gamma IFN , which Is labile at pH 2 . They have a
molecular weight o f about 17,000* are nondialysoble
and TinnseLlimentable (iQOjOOO Thcv WC poorlv
an tigen ic, so no routine serological tests arc
available for th e ir d etectio n and estim ation .
Interferon assay is based on its biological activity
such as the ability to inhibit plaque formation by a
sensitive virus. The potency oflETN is expressed as
International Units (1U) per ml.
Many properties o f interferon make it an ideal
candidate for use in the prophylaxis and treatment
o f viral infections; it is nontovic* nonancigenic,
diffuses freely in the body and has a wide spectrum
o f antiviral activity, The major drawback "initially
was its species specificity - interferon produced by
nonhuman cells was nor clinically useful. This was
overcome to some extent by producing interferon
from bully coat leucocytes from blood banks* with
N D V or Sendai virus as the inducer. Now, human
interferon is available in unlim ited quantities
following its commercial production by cloning in
bacteria and yeast. Even so, its initial promise as an
antiviral agen t has not been fulfilled. L o cal
application o f high doses has shown some benefit
against upper respiratory infections* herpetic
keratitis and genital warts. Limited success has also
been reported against generalised herpes infection
in immunocompromised hosts* and against hepatitis
J-] and C infections. Some encouraging results have
455
been reported in che use o f in terferon as an
anticancer agent* particularly in lymphomas but
there have been reports o f toxic effects in cancer
patients given high doses o f interferon.
Although interferon was first recognised as an
ant iviial agent; it is now known to be a more general
regu latory peptide belonging to th e class o f
cytokines. The main biological effects o f interferons
are the following:
1, A ntiviral effects: Induction o f km stance to
infectinns-
2 , Antimicrobial effects: Rcs-istance to intrjcclluiar
infections, for example toxoplasma, chkmydi-ii,
malaria.
3, Cellular effects: Inhibition o f cell growth and
prolife ration: and o f D N A and protein synthes is;
increased expression o f M H C antigens on cell
surfaces,
4, Immunoregulatoiy effects: Enhanced cytotoxic
activity o f NK* K and T cells] activation o f
macrophage cytwidal activity; moduiarion of
antibody formation; activation o f suppressor T
cells; suppression o f D T H .
L a bo rato ry D ia g n o s e s o f V i r a l
I!) [SI: ASKS
Technical d ifficu lties in virus iso latio n and
identifies ri,on> the length o f time required for these
procedures and the lack o f specific therapy for viral
infections have contributed to the sparse use o f
diagnostic virology till recently. T h e situation has
changed in recent ycair. W ith the development of
rapid techniques for the diagnosis o f many vltus
infections and the availability o f specific drugs
against at least a few viruses, diagnostic virology is
fast becoming a routine procedure.
The demonstration o f viral infection in selected
groups o f persons (screen in g) i,s an im portant
procedure in the prevention of some diseases (such
as screening for H E V and H IV in blood donors).
E tin logical diagnosis of viral Infections is useful in
many ways. It is o f vital important in some cases* as
in mbelL in pregnant women. It helps institution
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o f early Specific therapy us in herpetic encephalitis
and Ii2 sionR o f the eye. [r scras ro define the etiology
of vigue syndromes such as upper respiratory
iofectiOD or aseptic meningitis. It is essential for
rhe detection and prediction of epidemics and the
identification of antigenic variation in viruses. It is
invaluable in the prompt control o f outbreaks. !r
may Lead to the discovery of new viral infections.
Successful diagnosis o f viral injections dej>end
as much on the awareness (it rhe physician as on
the cap ab ility of the virus lab o rato ry . T h e
appropriate specimens should he collected from
patients-, preserved and transported to the laboratory
in the proper manner along with pertinent clinical
and cpidemioloiric.il information [Table 4L
A1).
In the llb on toiy, the following methods are
commonly employed; microscopic demonstration
o f the virus or its inclusion body, demonstration of
the vim s n otion , isolation and identification o f the
virus, or detection o f the specific antibody.

Respiratory system Throat swabh Nasopharvngeal aspirate {IF} Paired te n

nasopharyngeal thruur washing (EM )
upiniK t

Central nervous sysiem Feces, blood Brain biopsy (IF i t EMk Paired sera
{for arbovirus isolation) CSF (EM flt IF)
C S F (brain biopsy
throat swab, rectal sw.ihl

Cardiovascular system Feces Nil Paired sera

Skin Maculai/papular scrapings, Vesicular/puitullT fluid (EM fldD) PaiTtd sera
vesicular/pustular lluid, Ulcer tcrapinp (EM),
ulcer scrapings, emst, feces, ousts, [EM i t ID)
throar -swab

Eye Cunjunvlival scrapings Conjunctival scrapings, as smears Paired sera

or swabs on mietfMrtpe slides (LM St IF}1
Liver Blood (for yellow fever) Scrum [feces) Serum
General; congenital Throat swab
intectiuns (products of cQnceptkm) Nil Single sera
(rowdier St baby)
1 .'neraJ; PUC) Hcparinised blond (arbovirus Nil Paired sera
and arena,virus infections)
throat swabs,
feces (fresh urine)
'Specimens within brackets are not appropriate for routine diagnosis but may be indicated in particular
circumstances
Z1F = Immunofiuuitjxvence; EM - Electron Microscopy. LU ■ Immunodiffusion; LM = Light Micmstopy
'For diagnosis of rabies only
{Adapted bom W1 JO)

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 T h e d e m o n stra tio n gf virus
elementary bodies by examination o f stained smears
is nnw seldom employed. The detection o f vims by
electron, microscopy is being used increasingly. In
some diseases, it used to be the only diagnostic
method (for example viral diarrhea). Demonstration
o f the inclusion body is a routine diagnostic method
for rabies in dogs. The microscopic diagnosis ol
rabies has been rendered very sensitive by
fluorescent antibody techniques. 'The use o f direct
and indirect fluorescent antibody techniques for the
examination o f materia] from lesions, as well as for
the early demonstration of viral antigen in tissue
cultures inoculated with specimens has enlarged
the scope and greatly increased the speed o f virus
diagnosis.
I n tfcCH
where virus antigen is abundant in the lesions, its
dem onstration by serological me thuds such as
precipitation in gel or immunofluorescence offers
a rapid method o f diagnosis. H ighly sensitive
sero lo g ical tests such an court teri m mu un­
cle ctrophoresis, radioimmunoassay and en/vir.e
linked im m unosorbent assay have found wide
application in diagnostic virology ior the detection
o f viral antigens in clinical samples. M olecular
methods such as probes and pnlvmerase chain
reaction provide rapid, sensitive and specific
information about the presence o f viruses in clinical
samples. They arc fast becoming routine diagnostic
methods in the affluent countries, hut in the poor
nations, their avaitabilily is limited.
Fof virus isolation it is
imperative that the specimen be collected properly
and transported with least delay To the laboratory.
As moat viruses are heat labile, refrigeration is
essential during transport. The methods used tot
isolation depend on the virus sought. In general,
they' consist o f inoculation into animals, eggs or
tissue culture, after the specimen is processed to
remove bacterial contam inants. The isolates are
Identified hv neutralisation or Other Suitable
serological procedures. It has to he emphasised that
the mere recovery' o f a virus from a patient tk»cs
not justify the assumption that it is the causative
agent nf the patient’s illness. Many viruses [For
example adenoviruses, enteroviruses) arc frequently
found in normal individuals. The results o f isolation
should always be interpreted in the light of the
clinical data. Demonstration flf an immunological
response to the virus iso-late in tlie patient during
the course o f the disease reinforccs the significance
o f the isolation.
The dem onstration oi
□ ri--c in titre of antibodies to a virus during the
course o f a disease is strong evidence that it is the
etiological agent. For this, it is essential to examine
paired s t r i, the 'acute' sam ple collected early in
the course o f the disease and the 'convalescent'
sample collected 1 0 -1 4 days later. Examination ot
a single sample o f serum for antibodies may not be
m eaningtul except when IgM specific tests- arc
done. The serological techniques employed would
depend on the virus but those in general use arc
neutralisation, complement fixation, E l -ISA and
hem agglutination inhibition tests.
"I’llo availab ility o f
molecular methods has transformed the diagnosis
oJ viral diseases, cidarging the scope, sensitivity and
specificity of such tests.
Prolonged and effective immunity is a characteristic
o f most viral infections. Viral vaccines also confer
solid protection and are, in general, morr effective
than bacrerial vaccines. Viral vaccines may be live
or killed fTable 4 9 -2 ). Live vaccines are more
effective [Iran killed vaccines. Tlie smallpox vaccine
has been used as the sole tool for [he global
eradication o f the disease. The early live vaccines
were developed empirically from natural viruses (as
Jcnncr's c w p o s vaccine) or by attenuation by serial
passage (as yellow fever vaccines). The basis o f the
latter technique was ail unconscious selection of
avimlcnt mutant*- W ith the development o f more
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 RibomyelitLE Live AvlcuK 11 r strains grown in monkey kidney cell culture
Killed Virulent attains grown in monkey kidney cel] cutture,
formalin-killed
Rabies Killed (Semple type) Fixed vims ptijwn in sheep brain and inactivated by
phenol or beta propioLactorrt
Killed Virus grown in cell culture and inactivated with beta
pnopiolacfune
Yellow fever Live (I7D ) Attenuated virus grown in chick embryos and lyophilised
Japanese Killed Vims grown in mouse brain and inactivated by formalin
encephalitis
Varicella Live Attenuated virus grown in chick embryo fibroblastculture
Mumps Live Attenuated virus grown in human diploid Cell Culture
Influenza Killed (subunit) Virus disintegrated wiih sodium deoxycholaTt
Live (attenuated) Virus attenuated hy serial passage in eggs
Live (mutant] ts mutants which are y,virulent
Live (McombinanO Recombinants with surface antigens of new strains and
growth characters of established strains
Measles Live Attenuated vims grown in tissue culture
Rubella Live Attenuated virus grown m tissue culture
Hepatitis B Cloned subunit HBsAg cloned m yeast

precise genetic techniques, live vaccines have been
developed by plaque selection (Sabin vaccine for
p o lio m y elitis) o r from rs m u tan ts o r by
recombination (as in influenza).
Killed, vaccines have been prepared by
inactivating viruses with heat, phenol, formalin or
beta propiolacione. Ultraviolet irradiation is not
satisfactory because of the risk o f multiplicity
reactivation. The reactogenicity o f killed vaccines
has been attempted To be reduced by the purification
o f the viruses. Adverse reactions may also be
reduced by the use of'subunit vaccines' in which
the virus is split by determents or other chemicals
and only the relevant antigens Incorporated it] the
vaccine, Vaccine production by cloning the desired
antigen in bacteria or yeast is becoming increasingly
common, as in hepatitis 0 .
Live vaccines have the following advantages: A
single dose is usually sufficient. T h ey can be
administered by the route o f natural infccrinn so
that local i m munity is induced- They i nduce a wide
spectrum of Immunoglobulins to the whole range
o f viral antigens. They also induce cell mediated
immunity'- They provide more effective and more
lasting immunity than killed vlLCints. They can, in
general, be prepared inure econom ically and
administered more conveniently especially for mass
im m unisation. Som e o f them can be given as
com b in ed v accin es (m easles-m u m p s^ ru b ella
vaccine).
They have the following disadvantages: There
is a risk, however remote, o f reversion to virulen«-
T h c vaccine may be contaminated with potentially
dangerous viruses or other infectious agents. The
vims may spread from the vaccines to contact?.
While this is a serious danger in some situations,
as when spread occurs to unmunodeficietit or other
high risk contacts, in other cases, it may even be an
advantage (as in poliomyelitis where the range of
vaccination is extended by the natural spread o f the
vaccin e virus am ong ch ild ren and ad u lts).
Interference by pre-existing viruses may sometimes
prevent a good immune response following live

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Hidden page
460 h Itaxtboofc or Microbiology *

own thymidine kinase (IfSV , V -£ ) are for more F n sca rn ct ( tria n d iu m p h o s jT ii& n c y fn rin a te )

susceptible than those winch do not (C M V -E B V ),
The related drug t Ganciclovir is mure active against
CM V .
T h e widely publicised drug A zidorhym idinc
(Zidovudine, A Z T ) used against IIIV infection is
a thymidine analogue which blocks the synthesis
o f p ro v irii D N A by inhibiting '■".ral reverse
transcriptase. A Z T is used widely in H IV infection,
hui is toxic and costly.
A ierics (it dideoxynucleosides 1 DnfajlOiijie,
ZaJcr/abifie, Sfavirui'.'ie, Lamjvudj'nc) have been
svnthesi-ed and found in possess anti-HIV activity
by blocking reverse tra nsc ri |irasc. The second group
o f drugs uset I in H l\ uifeci ior- is protease inl 111v iors
(5arjujjMVjrh /Jifo-jravin Indinavir).
Jlifwvj'mj (Viraao/e) is a synthetic nucleoside
related tn guanosme. It shows activity against many
DNA and RNA viruses. Admin isteied as in
aerosol, it has been effective in the treatment of
respiratory syncytial viral infection and also in
inlluena. Intravenous ribavirin has been reported, to
be effect ive agai nst Lass* fever and other hemorrhagic
fevers.
O t h e r d rU jS t Amantacfrne fi4damanfj.ni.ni/n r
h y d r o c h lo rid e , S y m m e t r e l) blocks h o st cell
penetration bv influenza A virus but nor B or C . A
derivative rim zntad:ne is less toxic and et|ually
effective. A second line o f drugs against influenza
employs neuraminidase inhibition.
Enviroxinc and related chemicals have shown
activity ag.nnsc rhinoviruses.
specifically inhibits D N A polymerase o f the herpes
simplex virus and has some effect against hepatitis
B and H IV also.
Suramin developed as an anti parasitic drug in
1916 was found to inhibit reverse transcriptase
act hi tv and so was one o f the first 11m gs u-e Uaga ilist
AIDS- Because o f toxicity and inadecpiate efficacy
its use was discontinued.
I n t e r f e r o n s The discovery of interferons, with
activity agginst a wide range o f viruses raised hopes
of their application as antiviral drags. However,
most trials have been unsuccessful. Benefici.il effect
has hcen obtained in persistent injections such. as
hepatitis B and C, laryngeal papilloma and against
CM V infections in transplant recipients. High doses
of interferon lead to toxic effects.
In spite o f intensive efforts, progress in the field
cif antivi ral ch lj: i ii?Clit;rapy has not been ^.Lt isfietory.
Vjrious (actors contribute to this. Many compounds
show antiviral activity in tissue Culture hut most of
them prove to be ineffective or toxic in animal tecta.
The available drags have a narrow range o f activity.
They are seldom able to eradicate the virus from
the host so recurrence is com mon. Viruses develop
resistance to the drugs and break through infection
takes place even during treatm en t. The A ID S
pandemic has been a catalyst in development gf
an tiretro v iral drugs. It is hoped th at b e tte r
understanding of the molecular and cellular biology
of viruses and o f virus-host interactions may lead
to the development o f mote effective antiviral agents.

F u r th e r Reading
Collier L and_f Oxfoid .2000. H v m in V irolog y . London: Oxford Univn-ity Press.
Fields DN el al. 19%, FrroJq^v edn. Philadelphia Lippmcote- Raven.
Harper L3R. 1WJ. M iA eathr Oxford^ BioF.
Kuckerman AJ e( all 2000. CJimcaf VTraJqgy, +* cd. Qucbeiterjofon Wiley.

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 Bacteriophages
Bacteriophages (commonly abbreviated a> phages) are
viruses tliat infect bacteria. Twort (1915) described
degenerative change in staphylococcal colonics
isolated from calflynnph* which could be transmitted
serially by application o f culture filtrates from the
original growth. d'Herelle (1917) observed that
filtrates o f feces cultures from dysentery patients
induced transmissible lysis of a broth culture o f a
dysentery bacillus. H e suggested that the lytic agent
was a virus and gave ir the name bacteriophage.
P h ag es o ccu r widely in n ature in d o s e
li association with hacEerij. T h ey can be readily
isolated from feccs> sewage and other rtanir J sources
o f mixed bacterial growth. Early hopes that phages
could be used in the treatment o f bacterial infections
have nut been fulfilled but these viruses have
Contributed much to microbiology. As phages could
be grown easily on bacterial cultures, they provided
the only convenient model for the study o f vinis-
hosE interactions at the cellular and molecular levels
PHASES
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■. j.- -j •: t

before The development nf cell culture techniques bacterial genome, replicating synchronously with
made similar Studies with animal vtro*es ^mshhihlr:. it, causing no harm to the host cell (Fig. 5 i1 j ),
Phagts play an important role in the transmission . Replication o f a virulent phage can
o f genetic information between bacteria bv rhe be considered in the following stages adsorption,
prtm-csk of transducticu]. The present* o f phage p en etration , synthesis o f phage com p on en ts,
genome integrated with bacterial chromosomes assembly, maturation and release o f progeny phage
COnftrt on bacteria certain properties by a process particles.
known as philgc COnvttsktT). Hi ages have been used Phage particles Come lUTO Contact with bacLcriil
ns cloning vectors in genetic manipulations. TTic cells by random collision, A phage attaches to the
presence ot high ocincciirnttions of phage part ides, surface o f a susceptible bacterium by its tail.
upio 1 0 E per ml in some natural waters suggests Adsorption is a specific process anti depends -no
that they mu- have a role in the control o f bacterial The presence o f complementary chemical groups
populations in such environments. The specificity on rhe reccrtnr sites o f the bacterial surface and on
(it the host range (1I phages is the ba-sis ut ph;lge the terminal base plate o f the phage. Under optimal
typing methods, by which bacteria L-ar he identified c(mJ][ionv. adsorption it j very rapid process, being
jo J typed. complete within minutes. Certain tofeclnrs, such
■ Certain hactLTinphagvs that infect as cations, are necessary for adsorption. T h e
E , c o li called the T even phages ( T 2 , T 4 , Tfi),
have been studied in great detail and traditionally
serve as the prototypes in describing the properties
o f bacteriophages.
T even phages have a complex and characrcristie
morphology- T h ey are tadpole shaped, with a
hexagonal head and a cylindrical tail. The head
consists o f a rightly packed core o f nucleic acid
(double stranded D NA) surrounded by a protein
gpat or capsid. The size of rhe head Varies in
different phages from 2H mtt to HM1 n m .T h e rail is
composed o f a hollow core, n eon tractile sheath
surrounding rhe core and a terminal bate plate
which has attached to it prongs, mil fibers or both
(F ig . 5 0.1),
Though m ost b acterio p h ag es have the
morphology and structure described j Ijovc* phage*
char are spherical or filamentous and possess
single stranded D N A or R N A have also been
Identified.
Phages exhibit two different types
o f lifecycle. In tile viru len t or ly ric cycle,
intracellular multiplication tjf the phage culminate*
In the Ivsri o f rhe host bacterium and the release of'
progeny vifions. In the troTpcrafc or tysogenk cy d e
the phage D N A becom es integrated with the

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 1 BactMtaphageiii: ► 463

Fig. 50.3 lyUc and tyngonte ilfn ol im M qpIg i A- K teffdon, B, Injection id phage QfU, C, drcuterleaion of
phage ONA, 0. raplcatlon of phage DMA, IE, production rt phage component^ F. m — lMy of phage,
0. rokaro of progenyphage, ML Integration of phage DMAtrtfi hoet chromoeomo, LUnary ftrofon of lyoegeUe
bacterium, J. daughter bacteria carrying prophage, 1C i of prophage, L iiit h stage at C. {A to C
bucterial recepU&r rices may be situated in dltferent
layers o f the cell wall or on surface structures (such
as the V] antigen o f the typhoid bacillus} or
appendages (such as flagella or se* pill). Bacteria]
pro top lasts, w h ich are devoid o f eel] wall
components, cannot adsorb phages and therefore
will not be infected, H ost specificity of phages is
determined at the level of adsorption.. Experimental
infection by direct injection o f phage D N A can be
achieved even in b acterial strain s th at are
insusceptible to infection by the wliole phage. The
infection o f a bacterium by the naked phage lUutleic
acid is known as tntrtsfecrjofl.
Adsorption is followed by the penetration ot the
phage nucleic acid into rhe bacterial cell. The
process o f penetration resembles injection through
a syringe. T h e base plate and tail fibres are held
firmly against tire cell causing the hollow core fo
pierce through the cell wall. The contractile tail
sheath acts like a muscle imd derives its energy
try^nl _i ’itT"]m amount o f adenosine triphosphate
present on the tail of the phage. The phage DNA
is injected into the bacterial body through the
hollow core. Penetration may be facilitated by the
presence on the phage tail of lysozyme which
produces a hole on the bacterial wall for the entry
o f the phage core. The complex structure of the
phage particle is required only n>r the injection of
the nucleic acid into the hint celL '["he phage D N A
alone is necessary lot the initiation o f the synthesis
o f daughter phages. Alter ;wnetration, the empty
head and tail o f the phage remain outside the
bacterium as the shell oj 'ghost'.
W hen bacteria arc mixed with phage panicles
at high multiplicity (that is very large number of
phages per bacterial cell), m ultiple holes are
produced on the cell with the consctnimt leakage
o f cell contents, B acreri.il lyisis occurs without viral
multiplication.This is knowna s 'lysis from without’
(Fig. 5 0 .2 ).
Im m ediately after penetration o f the phage
nucleic acid, the synthesis o f the phage components
is. initiated- The first products to lie synthesised
(called c & i} y p r o t e in s ) are the enzymes necessary
for the building o f the complex molecule* peculiar
to rhe phage. Subsequently, i.itv protein? appear,
which include the protein subunits o f the phage
head and tail. During this period, the synthesis of
bacterial protein, D N A and R N A crates.
Pb.Lgc D NA, head protein and tail protein are
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synthesiseJ separately in the bacterial cell. The
DMA is condensed into a compact polyhedron and
'packaged' into the head and., finally* the tail
structures arc added- This assembly o f the phage
components into the mature infective phage particle
is known as [mentation.
Release of the mature progeny phages typically
occurs by lysis of the bacterial cell. D uring the
replication of the phage* the bacterial cell wall is
weakened and it assumes a spherical shape. Phage
emeyrttes act on the weakened cell wall causing it
to burst or lyse resulting in the release of mature
daughter phages.
The interval between the entry ot the phage
n u cleic acid into th e b acterial cell and the
appearance of the first Infectious intracellular phage
particle is known as the eclipse p h a se. It represents
the rime required for the synthesis of the phage
components and their assembly into mature phage
particles. The interval between the infection o f a
bacterial cell and the first release o f infectious phage
particles is known as the kterit period. Immediately
following the latent peKotin the number (it phage
particles released increases for a tew minutes till
the m axim um num ber of daughter phages is
attained. This period, during which the number of
infectious phages released rises, is known as the
rise period. The average yield o f progeny phages
per infected bacterial cell is known as the hursl
size. Th is is estimated bv experiments in which
in fection is established with o r e phage per
bacterium and the release o f infected phage particles
is estimated serialIv over a period o f time. The
results o f sweh an experiment plotted on a graph is
known as the one-step growth curve (Fig. 50.4).
Unlike virulent phages which
produce lysis etf the host Cell, temperate phages
enter into a svmbinric relationship with their host
cells without destroying them. Following entry into
the host cell, the temperate phage nucleic acid
becomes integrated with the bacterial chromosome.
The integrated phage nucleic acid is known as the
jiropha^e. The prophage behaves like a segment o f
the host chromosome and replicates synchronously
with it. This phenomenon is called lysogeny and a
bacte ri um that carries a prophage with in irs ge nnm e
is called a h'sogtnic b icterium. I,ysogenisaiLon docs
not upset the bacteri.il metabolism.
The prophage confers cert sin new properties
liii the lysogenic bacterium . This is known as
Jisojjerjjc ccm-efision or p h a ge corners tart- This is
due to the st nrhesis o f new proteins that aie coded
tor by the prophage D NA. An esimple is toxin
production by the diphtheria bacillus, which is
determined by the presence in It of the prophage
beM. The elimination o f the prophage abolishes
the toxigenicity o f the bacillus.
During the multiplication of lysogenic bacteria*
the prophage may become 'excised’ from occasional
c e l l The excised prophage initiates lytic replication
and the daughter phage particles arc released,
which in toe t other bacterial cells and render them
lysogenic. This is known a> spontaneous induction
o f prophage, While this is a ru e event, all lysogenic
bacteria in a population can be induced to shift to
the lyric cycle by exposure to certain physical and
chemical agents. Such inducing agents include UV
rays, hydrogen peroxide and nitrogen mustard..
A lysogenic bacterium is resist.mt to reinfection
by rhe same or related phages. This is known as
s u p e r in fe c t io n iin n w n iiy .
Bacteriophages may act us carriers of genes from
one bacterium to another. T h is is known as
transducrion. Tw o types o f tran sd uction are
recognised- In restricted transduction, only bacterial
genes contiguous to the prophage are transmit ted.
For example, transduction by ihe prophage furotait
in E . COii K 1 2 tran sfers only th e ga1J gene
(determining termentation o f galactose), which is
the bacterial gene contiguous to the prophage. On
the other hand, any bacterid gene may he transferred
in generalised transduction. Transduction has beer
dem onstrated in -many gencm o f bacteria and
const! Kites one of the most important mechanisms
o f genetic exchange among bacteria in nature.
Plasmid mediated drug resistance in staphylococci
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4 6 6 * Textbook ol Microbiology *■
in an example of a medically important property
that is transmitted by transduction.
Phage par titles exhibit general 11■' y o f type
and a low rate o f heritable vari.inop.
If i bacterium si multaneously adsorbs two
related but slightly different DNA phage panicles,
both can infect and reproduce. O r lysis both types
are released. W hen this occurs many o f the progeny
are observed to be recombinants.
P h a g t; A s s a y
W hen a phage 'L- applied on the lawn culture of a.
susceptible bacterium, areas o f clearing occur after
numbati- in. These zones o f ly'-is are called piaquCS.
rb e size, shape and nature o f plagues are
characteristic for different phages. Since under
optim um conditions a single phage particle is
capable o f producing one plaque, plaque assay can
be employed for titrating the number o f liable
phages in a preparation. As plaques are analogous
ti i bjiclt'T i■lI colonies, plaqumg is aLsn useful for the
purification of phages.
P hage T yhjmcj
Tile specificity uf phage-bacceriuin interaction ls
made use o f :.n rh* identification and typing of
bacteria. Phages exhibit different degrees o f host
specificity. Some phages possess wide host ranges,
covering many bacterial genera, while others have
a narrow range lim. ited to certain Rtr.i ins o f bacteria
only. W ith some phages serial passage in a strain
o f bacterium makes them spe. Iflc for that strain
and related strains (adaptation o f host range).
Phages .ixu available that lyse .ill members of a
b acterial genuR (fo r exam ple genuR-Rpcci fid
bacienophage for SdmoneJila), ad members o f a
species (for example specific bacteriophage (dr ff.
j jr f h r a c H , and all m em bers o f a biotype or
subspecies (for example Mukerjee's phage IV which
lyses all strains of classical V. t h o le n e but not V.
cfui terse hiotype E l Tor). T h e most important
application o f phage typing is for intraspecics typing
o f bacteria, as in the phage typing o f if. typhi and
staphylococci. Adapted phages, active only against
fresh isolates possessing the Vi antigen, are used
for phage typing o f typhoid baciin. Staphylococcal
phage typing is a pattern method, using a set of
standard phages. A str.un o f Staphylococcus may
be lysed by a number o f phages and the phage type
of a strain ls designated bv the numbers o f the
different phages that lyse it.
As lysis is influenced bv the dose o f infection,
phage preparations used for typing should be
standardised by titration.Titration is carried out by
applying - ltl.hI dilutions o f the phage preparation
on a lawn culture o f a susceptible strain and
observing the ]y>is after incubation. The highest
di Iutiun o f the phage prepara!iim that IList produccE
confluent ly^is i>. known as the routine test dose
(R T D ).
BACTE R(OC(N9
Gratia (1 9 2 5 ) observed the production o f a highly
specific antibiotic substance hy one stiai:i o f £ . cots
which was active against another strain of the same
species, T h e name colic in was given to such
Ruhstancefi produced by E . c«fiJ and other members
o f rhe fam ily E n tc r o b a c te r k c a c . W ith the
r c c o g n irio r th at c o licin -lik c su b stan ces arc
produced by several other bacteria also, the generic
name baeteWocin was proposed for the group o f
liighly specific antibiotic-like substances produced
by certai ilstra ins o f bacter uwli ich are active agai nst
other htrail ns of the same or different sp eck s.
Bacteriocins are giver specific names based on the
bacter iid spec ies o f origin, for example cube ios from
E .tali, pyocins from JV pycycy^ncx (.a-ns^ino.-st),
mcgacins from B, m egs ten um and diphthcricins
from C . diphrhesi-je.
Bacteriocins are prolei [is but some may have
asset iated lipopolysatthstrides derived from the cell
walls o f bacten i producing them. Bactentk-ins and
phages resemble each other in a number o f respects.
Both adsorb o r the surface o f susceptible bacterid
cells on specific receptor sites some of which may
he the same for phages and bacteriocins, Under
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rhc electron m icro sco p e, some b acteriociuE,
especially proems, appear like [Ire rail structures of
phages. 1 hey may be considered products o f
defective phage genomes, able to emir only ft>r parts
o f phage particles.
The synthesis o f bacterimuns is (ieEuTiTiiuud 1>V
rhe presence in bacteria of eolidnogen.it: [actors
'.1
Anderson "IT. 1981. Reflections on phage genetics. Ann Rev-Gfricrrcs 15:^05
Dmv M. 199S. Baaenocins and bacteriophages, I il TopJey & IVifsOTaJl .^'crDfrtikgpt Arid Mo-robin f In fsedooi, Vul.2.
J.ondon: Arnold.
Mithcm CK.et al teds) 1983 filrtEnspIlfe TJ. WwhiJlgtrrn. J>-C: American Sodety hir Mitcobiolc^y.
tCol factors), Col factors are cpisomes and can be
Tran emitted from cell to cell by conjugation or
transduction, Certain physical and chemical agents
(U V raya, nirrogen m u stard } induce co h cin
production by the cells harbouring Col factors.
A cell producing a bacteri.ocin is immune to i[
hut m ay be sensitive to o th er b a c tcrio c in s.
Bactcriocins have a Voty specific activity on bacteria,
being capable o f killing some b»T not all strains of
j species. The specificilv :s made use o f in rvying
certain species such as Sh. Satina, Proteus up., f t .
aerugjnu.TrJ. H acteriocins kit] susceptible cells
without lysing them.
W hile phage typing schemes are generally bused
on the sensitivity of the test strains to the lytic action
o f phages, bacteriocin typing schemes depend on
th e abi ILty of bactcrioci ns produced by the test strai n
to kill standard indicator strains o f bacteria. T h e
usual method of haeteriiodn typing employs ike
plate diffusion technique. The lest bacterium is
inoculated as a brood streak on the centre ni a
culture medium, the bacterial growth is scraped
off and the remaining cells killed by exposure to
chloroform vapour. Standard indicator strain of
bacteria art; then Streaked at right angles TO the
original inoculum. After incubation, the pattern oE'
inhibition o f the indicator strains represents the
bacteritHiir type of the lest bacterium (Fig. 5 0.5).
. _■ r ..... ’
I
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unique in than it lh .lei H
aiti(icia] virus" jiu .1 does
occurin nature is such. ][ hasbeen studied m greater
detail than variola, as it is safer to work with.
Vaccinia Vitus is being employed as a Vector fur
the developm ent o f recom binant vaccines. T h e
vaccinia genome can accommodate about 25,0 0 0
foreign base pairs sufficient for introducing several
genes. M a n y genes have been inserted, including
[hose coding for rhe antigens o f hepatitis Q virus,
H I V , rabies, ami for pharm acologically im p jrtan t
p ro d ucts such as neuropeptides. H o w e v e r the
vaccinia vims is not suitable as a vector for human
use due to its pathogenic effects.
V accinia and variola viruses are so sim ilar in
their properties that they can be considered together.
T b e v irio n is b rick shaped, in
vertical section it consists o f a double layered
membrane w hich surrounds a biconcave 'nucleoid1
containing the D N A core. O n either side o f the
nucleoid is a Lens shaped structure called rhe lateral
body (F ig , 5 1 . 1 ) r T h c virion measures about 30Q *
200 * 10 0 nm and so can be seen under the light
m icroscope. Variola virus was first demon strafed
m icroscopically hv R u isl in 1S S 7. Paschen in I90fi
developed a staining technique for the virus particles
and demonstrated rhe elementary bodies (Paschen
bodies) in smears from smallpox lesions.
.c t t y i'.'■ H.1 a n d o n e in ic a J p f o p c r t i^ l!
Poxviruses arc stable and if protected from sunlight
m ay remain viable for months at room temperature.
In the cold or when freeze dried, they survive for
years. T h e y arc susceptible to ultraviolet lig ht and
other irradiations. T h e y ate resistant to 5 0 % glycerol
and 1 % p h e n o l but arc re a d ily in activ a te d by
fo rm alin and o xidising disinfectants. T h e virion
consists essentially o f D N A , protein and lip id .
T h o u g h etivelnped, the virus is not inactivated by-
ether. T h e virion contains a m ultiplicity o f enzymes.
T h e entire m ultiplication o f the virus takes place
in the cytoplasm o f the infected cell.
Alt p o jtv i runes share a
c o m m o n n u c le o p r o tc in ( N T ) a n tig e n . B y
im m unodiffusion some twenty different antigens
not
I ran g n j v it e o n c M 'M of H it c m n t two i 0
b e $ t|g ( t l j c t i l t irtrto n ife is ritH n •
tthloh tm m Hntfltr turbo*.
have been identified. These include the L $ antigen
(a complex o f two antigens, the h e it labile h and
the heat stable S- lanrigens), agglutinogen, and
h e m a g g lu tin in , w h ich is re sp o n sib le for the
agglutination o f erythrocytes o f those fowls w hich
aie also agglutinated non suet ideally by tissue Lipids.
T h e v a rio la
and vaccinia viruses ran he differentiated hy their
growth characteristics and host range.
B o th viru se s gro w o n the
C A M o f 1 1 - 1 3 day old chick embryo producing
pocks in 4 8 -7 2 hours. Variola pocks are sm all, shiny,
w hite, convex, n o n -n e cro tic, n u n -h e m o rrh a g ic
lesions. V accinia pocks are Larger, irregular, flat,
g re y ish , n e cro tic le sio n s, som e o f w h ic h are
hemorrhagic (Fig. 5 1 .2 ) . T h e viruses may also be
differentiated by their 'ceiling temperatures', the
highest temperature above w hich pocks ate not
produced. 1 lie ceiling temperatures are 4 1.0 “C fqr
vaccinia, 3 8 "C for variola m ajor and 3 7 .5 ° C for
variola minor.
Variola anti vaccinia viruses can be
grown in tissue cultures o f m onkey kidney, H e L a
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and ch ic k em bryo cells. L’ ytopathic effects sre
produced by vaccinia in 2 4 -4 B hours and m un;
slowly by variola. E o sin o p h ilic inclusion bodies -
Cjuarm eri bodjes - can be demonstrated in stuncd
prep a ratio n s. T h e in c lu s io n bodies co n sist o f
annexations o f virus panicles in a matrix. Vaccinia
but not variola vim* produce!; plaques in c llic k
embryo tissue cultures,
' ; T h e vaccinia virus can i lifter a wide range
of animain experimentally. M onkeys, calves, sheep
and rabbits can be infected by scarification leading
Co vesicular leu Ions. T h e vario la virus produces
sim ilar lesions only in monkeys. Scarification o f
rabbit cornea w ith variola virus leads to keratitis
and se ctio n s o f the cornea w ill show typ ical
G u a rn ie ri h o d i« , In tran a?a1 in stilla tio n o f tbe
variola vim s in the m onkey produces a self-lim ited
attack o f smallpox with generalised skin lesions.
f .; . - 'i ::
Sm allpox has been eradicated.The last natural case
o f v ario la m a jo r detected was S a ib a n B ib i f a
Bangladeshi wom an found with smallpox un the
K arim gani railway platform in Assam on 24 \ta v ,
19 7S . T h e Last case o f variola m ino r occurred in
M crca, S o m a lia , in O cto ber 1 9 7 7 . T h e co m in g
f
generations are unlikely to witness the disease but
itv disappearance has been too recent for it to be
ignored altogether. A brief account o f sm all|iox is
therefore being prevented, (For more details about
smallpox, the 3 d edition of this textbook m ay he
consulted-)
Sm allpox was an exclusively human infection,
with no anim al reservtnr. T h e re were HO carriers
a ; the viintx was elim inated com pletely from the
patient on recovery. 'th e source o f infection was a
patient in the early phase y f the disease, though
injectivity extended from the appearance o f buccaJ
mucosa! lesions (enanthems) to the disappearance
nt all the skin lesion {exanthema). Infection usually
occurred only in cl-use contacts. Virus entered the
h(jdy by inhalation. After initial m ultiplication in
the local lym phoid tissues, the vim s reached the
r e t ic u lo e n d o t h e lia l c e ils , w here fu rth e r
m u ltip lic a tio n took place, le ad in g to a severe
viteEuia with seeding of (he mucosa and skin
heralding the clinical disease.rfh e incubation period
was around 1 2 days.
' I “he single crop o f centrifugal exanthems passed
through macular, papular, vesicular and pustular
stages, b efo re s c a b b in g and h e a lin g by scar
form ation i]t 2 - 4 weeks. T h e exanthems varied in
o p y rfo h le d m aterial
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 Herpesviruses
The: herpesvirus fam ily contains uVer a hundred
hpcci.es o f envelo p ed D M A viruses that: affect
hum ans and anim als. T h e y arc; characterised be­
thel r ability to establish latent infections, enabling
rhe virus to persist indefinitely w ithin infected hosts
and to undergo periodic reactivation.
T h e herpesvirus capsid is icosahedral, composed
o f 16 2 capsomers, and enclosing the core containing
the lin e ar double stranded I3N"A genom e. T h e
nucleocapsid ls surrounded hy the lipid envelope
d e riv e d from the m o d ifie d host c e ll n u cle a r
membrane through w hich the naked virions bud
dieting replication. The envelope carries surface
spikes, a b o u t? ntr long. Between the envelope :md
the capsid is an am orphous structure called the
te g u m e n t, c o n t a in in g several p ro te in s . T h e
enveloped virion measures about 200 run and the
naked virion about 10 0 nm in diameter.
Herpesviruses replicate in the host cell nucleus.
T h e y form L’ owdry type A intranuclear (Lipschure)
in clusio n bodies, L ik e other enveloped viruses,
herpesviruses arc susceptible to fat solvents like
alcohol, ether, chloroform and bile salts. 'I'hey arc
heat labile and have to be stored at - 7 0 ’ C .
T h e fam ily H eipesviridae is divided into three
s u b fa m ilie s based on b io lo g ica l, p h y sica l and
genetic properties:
A lp h a herpes viruses, w ith a relatively short
replicative cycle ( 1 2 - 1 S hours), a variable host
range and :l tendency to cause Intent infeed on in
se n so ry g a n g lia . In c u ltu re th e \ are ra p id ly
cytopathic and infectious viruses may be released
from ce lls, for exam ple herpes simples virus,
varicella-zoster virus.
ffentherpes^ruses, w hich replicate slowly (more
than 2 J hours), have a narrow liost range, grow
best in fibroblasts w ith a tendency In produce
enlargement of infected cells (cyromegaly) and cause
latent infection of salivary gland anti other organs.
In culture, the cytopathic effect is slow and the
v iru s re m a in s cell a s so cia te d , fo r ex a m p le
cytom egalovirus.
C7jr?]mahefjte.svrJTive.s, which have a narrow
host range, replicate in lym phoblastoid cells, are
specific for either li o r T lymphocytes and frequently
cause latent in fe ctio n in ly m p h o id tissue, for
exam ple Epstein -Ttarr virus.
V- ight differs n r types o f herpesviruses a rc known
whose prim ary hosts are humas. T h e y have been
officially designated ‘H um an herpesvirus types 1 -
H but their com m on nam es co ntinue to he in
general use, except for types 6 ,7 and S (Table 5 2 -1).
T h c herpesvirus fam ily has no com m on group
antigen and the different herpesvirus species do noi
show any significant antigenic cross reaction, excepL
between Herpes simplex types 1 and 2.
-i l • - L _ i l M f l
T h e her]ieH simplex virus (H S -V ) occurs naturally
only in hum as, but it can produce experimental
infection in many laboratory animals. There are two
types o f the herpes sim plex virus. H $ V type l
(H u m an herpes virus type 1 or H i I V type 1) is
usually isolated from lesions in and around rhe
m outh and is transm itted by direct contact or
droplet spread from cases or carrier*, H S V type 2
{ H H V type 2) is responsible for the m uiorily o f
g e n ita l h erp e s in fe c tio n s an d is c o m m o n ly
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 Human herpesvirus type 1 ] terpes simplex virus type 1 :i IjiJui
Human herpesvirus type 2 Herpes simplex virus rvpe 2 alpha
Human herpesvirus type 3 Varicella zoster virus
Human herpesvirus type 4 Epstein-EJarr vims
Human herpesvirus type v Cytomegalovirus
Human herpesvirus type 6 Human II ueU IvmphouiopiL: ben
virus*
Human herpesvirus type 7 R K virus*
Human herpesvirus type H
* These name* are no longer in use
transmitted Vertereilly, IiUfaCeHehral inoculation in
rabbits and mice leads to encephalitis. and corneal
scarification produces keratoconjunctivida in rabbits.
T ire 1 v im s grows in a v a rie ty o f p rim a ry and
continuous cell cultures (m onkey or rabbit kidney,
hu m an A m n io n , H e L a ) p ro d u cin g c y tu p a ih ic
changes, w ell defined foci w ith heaped up cells
and syncytia] or giant cell form ation. O n ch ic k
embryo C A M , small (diameter less titan 0.5 mm)
white shiny non-neurotic pricks are produced (Fig.
5 2 -2 ) - T h e fwo types o i the v iru s cross react
serologically. T h e y can be differentiated by the
following features:
1) A ntigenic differences can be made out using type
specific m onoclonal antibodies.
2) O n c h ic k em bryo C A M type 2 strains form
larger pocks resembling variola,
3) Types 2 strains replicate well in ch ick embryo
fibroblast cells, w hile type 1 strains do so poorly.
4) T h e in a c tiv ity o f type 2 is more temperature
sensitive than that o f type 1 -
cytolytic neultm*
p to ly tK neurons
alpha Cytolytic neurons
ifajnma Lvmphopreiliterativc lymphoid tissues
beta cytomegalic secretory
glands
Kidneys, other
organs and
tissue*
hinphoprolderarive lymphoid-1issues
beta lympfooprcliferaTive lymphoid tissues
pm m i
5) T y p e 2 stra in s .ire m ore n e u ru v iru le n t in
laboratory anim als than type 1.
6) Type 2 Strains are more resistant to antiviral
agents like I U D R and cvturahlne in culture.
7) Restriction endonuclease analysis o f viral D N A
enables differentiation between the [Wi> types
as well as between strains w ithin the same type.
H e rp e s sim plex is o ne o f the
mtrSt comm on viral Infections in humans* about 6 0 -
90 per cent of adults showing detectable antibody.
P rim a ry in fe ctio n is u su ally acq u ire d in early
c h ild h o o d , between tw o and five years o f age.
H u m u s are the only natural hosts and the sources
o f Infection arc saliva, skin lesions or respiratory
secretions- Asym ptom atic carriers form the more
important source or'inlectm nh especially in genital
infection with type 2 strains. Transm ission occurs
by close contact and may be venereal in genital
herpes. T h e virus enters- through defects in the skin
or mucoLLH membranes and multiplies locally; w ith
uell-to-cell spread. T h e virus enters cutaneous nerve
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Hidden page
area w ith w idespread u lee ration. A clinic: ally
in d is tin g u is h a b le p icture is also pro duced by
vaccinia vim s infection., both designated Kaposi's
vxriccUifornj eniptinn.
T h e buccal m ucosa is the site most
C o m m o n ly a fie c ted. G in g iv o s t o m a t it is a n il
pharyngitis, arc rhe most frequent conditions in
prim ary infection and recurrent herpes lablalis in
recurrent infection. T h e vesicles maV ulcerate and
become secondarily infected.
H S V infection is the most enmmon
cause o f corneal b lin d n e ss in som e developed
countries. Acute keratoconjunctivitis may occur by
itself or by extension from facial herpes- Follicular
conjunctivitis with vesicle formation on the lids is
another nna nitestati on. T h e cornea may be iinvolved,
w ith ty p ic a l b r a n c h in g d e n d r it ic u lce rs.
D ehri dement, Topical ant wind drugs ami iuterknnn
help in healing. Steroids are contraindicated as they
lead to deep stromal involvement and healing may
be delayed, with scarring and co nical blindness.
C ho rio retinitis and acute necrotising retinitis are
uncom m on but serious manifestations.
H S V encephalitis though rare, is
the most com m on sporadic acute viral encephalitis
in most parts o f the world. H S V encephalitis has
an acute onset, with fever and focal neurological
symptoms. B rain biopsy was employed in diagnosis
for instituting early specific therapy. T h is in now
replaced by demonstration o f H S V D N A in C S F
by P C k , w hich is a veiy sensitive rest in the acute
stage. H S V m eningitis is a self-lim iting disease,
usually resolving in about a week, w ithout sequelae.
T h e C S F shows lymphoeyrie pleocytosis and may
yield the vim s in culture.
H S V can cause sacral autonom ic dysfunction
and also rarely transverse myelitis or rhe G u illia n -
Barre syndrome. H S V has been im plicated in the
etiology nf Bell's palsy.
H S V esophagitis m ay cause dysphagia,
substcrnal pain and weight loss. It may involve the
respiratory tract causing tracheo bro nchitis and
p n e u m o n itis. H S V is an u n co m m o n cause o f
hepatitis. Erythem a m ulti forme m a t be seen in
association with H S V infection. Dissem inated H S V
in fe c tio n m ay o ccu r in p a tie n ts w ith
immunodeficiency, malnutrition or bum s.
In the 19 70 s genital herpes became the
most rapidly increasing venereal disease, particularly
in the U S A . In m en, the lesions occur m ainly on
the penis, or irk the urethra causing urethritis, in
women, die cervix, vagina, vulva and perineum are
at k it e d . W h e n o n ly rhe cervix is involved, the
in fe ctio n m ay be asym p tn m arie. The p rim ary
infection is usually more serious, accompanied by
system ic features like fever and m alaise. It is
followed by several recurrent episodes whieh arc
milder. T h e v csicu lo -ulcerative lesions may be very
p a in fu l. R e cta l and p erin e al lesio n s o ccu r in
homosexuals. Both types oi l I S V m ay cause genital
le sio n s, th o u g h H S V 2 is re sp o n sib le m ore
frequently and causes m any more recurrences.
There have been several reports o f an association
between .1IS V 2 and carcinom a o f the cervix uteri
but a causal relationship has not been established.
Coj^entru/r Transplacental infection with H S V 1
O r 2 can lend to congenital mall on nations. but N ils
is rare. Infection may occur during birth, particularly
if the mother has genital lesions due to H S V 2. In
such cases, cesarian section may prevent infection.
Pb&rnaial infection is more com m only due to
H S V 1. Neonatal herpes m ar be confined to [be
eyes, m onth o r skin, but is m ore co m m o n Iv a
d is s e m in a te d d isease h a v in g m u lt i-o r g a n
involvem ent, w ith oi w ithout encephalitis. T h e
mortal ifv rate is very high and survivors may have
neurological im pairm ent.
T h e diagnosis of herpes virus
ilifecrion m ay be made by microscopy, aurigen nr
U N A detection, virus isolation or serology.
T h e T za n ck smear is a rapid, fairly
sensitive and inexpensive diagnostic met bod. Smears
arc prepared from the lesions, preferably feom the
bane o f vesicles and stained w ith 1 % aqueous
so lu tio n o f to lu id in c blue 'O’ for 1 5 seconds.
M ultinudfcitfld giant cells w ith faceted nuclei and
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homogeneously R uined "ground glass" chrom atin
{ T z a n c k c e lls ) c o n s titu te a p o s itiv e sm ear.
Intranuclear type A inclusion bodies m ay he seen
in Giernsa stained smears. T h e virus particle may
aU o be d e m o n stra te d under the e le c tro n
m icro sco pe. It is not possible to differe ntiate
between herpes sim plex and varicella zoster by
m ic ro s c o p y T h e h erp e sviru s antigen m ay be
demonstrated in smears or sections from lesions by
the fluorescent antibi>dy technique.The fluorescent
antibrnly test on hrain biopsy specimertH provide*
reliable jo d speedy diagnosis in encephalitis. P C R
based D lV A detection has replaced brain biopsy.
Inoculation in mice and on chick
embryo C A M is insensitive and has been replaced
by tissue culture for virus isolation. Prim ary human
em bryonic kid n ey human am nion and many Other
cells are susceptible, but human diploid fibroblasts
are preferred. Vesicle fluid, spinal fluid, saliva and
swahs m ay he used. Typical cvtopalhic changes may
appear at early as in 2 4 -4 8 hours but cultures should
be observed for two weeks before being declared
negative. D ru g susceptibility too can be tested in
cell cultures,
Ditlcrentiarioo between H S V types 1 and 2 may
be made by a variety o f serological techniques, by
n u c le ic a cid h y b r id is a t io n or by r e s tric tio n
endonuclease cleavage and electrophoretic analysis
o f viral D N A or viral pnotiens.
Serological methods are useful in the
diagnosis o f prim ary infections. Antibodies develop
w ithin a tew days of infection and rise in title of
a n tib o d ie s m ay be d e m o n s (rated by E L I S A ,
neutralisation or com plem ent fixatio n tests. In
recurrent or reinfection herpes, there m av he little
change in the antibody titre,
Ido xyuridine U*ed topically in
eve and skin infections was one o f the first clinically
successful antiviral agents. T h e in tro d u ctio n o f
a cy clo v ir and v id a ra b ira enabled the effective
management o f deep arid *vsteniic infections. Early
treatment w ith intravenous aendovir lias improved
the outcome of encephalitis. Oral and topical use
m ay help in less, serious conditions. V alaciclovir
and fa m ciclo v ir are more e le c tiv e oral agents.
W h e n resistance to these drugs develop, drugs like
toscamct w hich arc independent o f viral thym idine
klnast action may be useful.
T h is virus was isolated by S ab ir and W rig h t (1^ 3 4 )
fro m the b ra in o f it la b o ra to ry w o rk e r w ho
developed fatal ascending myelitis after being bitten
by an apparently healthy monkey. It came to be
known as the B h virus from the initials o f this
patient. M a n y sim ilar cases have been reported since
then. Herpesvirus si m i ae i nfects old world monkeys
in the same m anner that herpes simplex intects
hum ans, the inferrion usually being asymptomatic.
T h e typical lesions produced are vesicles on the
buccal mucosa w hich ulcerate shedding the virus
and infecting contacts. Though most hum an C iieS
have followed monkey hites, the infection in some
was acquired through the h an d lin g of m onkey
tissues.
Herpesvirus sim ile is sim ilar to herpes simplex
virus in its properties. T h e two are antigenically
related but the herpes simplex virus antibody docs
not protect againsr herpesvirus sim iae infection. A
to n n u b -n l vaccine has been tried experimentally
in laboratory workers at risk.
T h e disease in hum ans is usually fatal. 'H ie rare
patients w ho survive have serious neurological
seq u e lae. T h e o ffic ia l nam e fo r R v iru s is
Cercopithcaine herpesvirus 1.
V - h IL r'L-l A A jA T A f
As early as 1 fifty. Von Bokay had suggested that
v a ric e lla (c h ic k e n p o x ) and herpes 7. 0 ster are
different manifestations o f the same virus infection,
Virotogical and epidem iological observations have
proved Thii. concept. T h e virus is therefore called
varicella roster virus ( V Z V ) . C hickenp o x follows
prim ary infection in 2 . nonim m unc individual, while
herpes zoster is a reactivation o f the latent virus
when (be im m unity has fallen to ineffective levels.
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 * Herpesviruses
T h u s , ch i. kenpuut caught1 bur m u luster. Contact
with either zoster or chickenpox may lead only to
clrickrnpox hut not zoster.
v z v is sim ilar to the herpes simplex virus in
its morphology. It does tint grow in experimental
an im a ls or c h ic k em bryos. T h e virus was first
isolated by W e lle r in hu m an eembryonic tissue
culture. It can be Brown in cultures o f human,
fibroblasts, hum an a m n io n or H e L a cells. T h e
cytoparhic effects are sim ilar to, bur less marked
than those produced by the herpes simplex virus.
In cultures the virus remains cell associated and
does not appear free in the m edium. B y using highly
specific antisera* it is ]*KS i :t1£ to distinguish k n i t i l
herpes virus types 1, 2 and varicella zoster viruses.
O n ly one antigenic type o f V Z V is known.
V a r i c e l l a ( c l iie k e n p n x ) : C h ic k e n pox is one
o f the m ildest and most com m on o f ch ild h o o d
infections. T h e disease may, however, occur at anv
age. A d u lt chickerpox, which is m are serious, is
rather comm on in some tropical areas.
T h e source o f infection is a eh ickenpav or herpes
zoster patient. Infectivitv is m axim um during the
ir trial stages o f the disease when the vim s is present
abundantly in the upper respiratory tract. T h e buccal
lesions w hikh appear in the early stage nfthe disease
and the veshnlar fluid are rich in vim s content.
Infcclihity wanes as the disease progresses and the
scabs are v irtu a lly n o n in fectious. T h e re arc no
anim al reservoirs o f varicella.
T h e portal o f entry o f the virus is rhe respiratory
tract or conjunctiva. A fter an incubation period o f
about two weeks (7—23 days) the lesions begin to
appear. T h e patient is Considered to be infections
during the two days before and five days after the
onset o f the lesions. In ch ild re n , there in little
prodromal illness and the disease is first noticed
when skin lesions appear. Buccal lesions may not
he noticed. T h e rash appears usually on the trunk.
T h e evolution o f the lash is so rapid that rhe various
stages - m acule, papule, vesicle, pustule and scab -
cannot be readily followed in individual lesions.
T h e rash is centripetal m disti ibution, affecting
t 479
m ainly the trunk and sparing the distal parts o f the
lim bs, and is very superficial without involving the
deeper layers o f the skin, resembling a dew drop
lying on the skin. The rash appeal in CTOpfi during
the first three tir four days o f the disease, so that
lesions o f varying age can be noticed on the same
patient. It matures very quickly, beginning co crust
within 4S hours.
W h e n varicella occurs in rhe adult, system ic
symptoms m ay be severe, the rash very profuse and
the entire disc use m u ch m ore intense th an in
children- T h e w h may become hemorrhagic and
occasionally bullous ksio n s appear. Pitted scars on
the sk in m ay re m a in after recovery. V a ric e lla
pneum onia is more com m on in adults, and often
fatal in the eld erly. O t h e r co m p lic a tio n s like
m yocardium , n e p h ritis, acute cerebellar ataxia,
m eningitis and encephalili-- may ensue. Secondary
bacterial infections, usually due to staphylococci
or streptococci, may occur. R e y syndrom e m ay
follow varifella in som e cases w ith a history o f
adm inistrano n o f salicylates. B u t in most cases,
chickenpo* is an uneventful disease and recuvtty is
the rule. O n e attack confers lasting immunity.
Chickenpox in pregnancy can be dangerous for
both m other and bahy. H ie disease tends to he mure
severe in pregnant women, with enhanced ri.sk o f
c o m p lic a tio n s like pneum om .L. T h e baby m ay
develop two types o f com plications, depending on
the period o f gestation when the w om an develops
chicken pox. I f maternal varicella occurs during
the first half o f pregnancy, the fetal i nfection may
usually be asymptomatic. Som e infants may develop
the fe ta l v a ric e lla sy n d ro m e, m a n ife s tin g as
c ic a t r is in g s k in le s io n s , h v p o p la s i .1 o f lim bs*
chorioretinitis and C N S delects. Some habies mav
not exhibit any delects, but may c a n y latent V Z V
infection. W h e n maternal varicella occurs neat
delivery babies may develop congenital ffleonacilj
varkreffa, wi thin two weeks o f b i rth. I f the mother's
rash began a week o r more before delivery, she
would have developed .mi diddle- w hich would have
been passed, along w iih the virus, to the fetus
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tram placenta Ily, btu."h a baby, though infected,
usually escapes c lin ic a l d ista n t. It the m other
develops chicken post les> the a week (or w ithin 2
duvs) ut delivery, the haby w ould have TL'ceived.
from the mothe r only the virus ami not the antibr>dv,
so that iT develops neonatal varicella. T h is is usually
a serious dissem inated disease, with high risk of
pneum onia and encephalitis. A s treatment for such
conditions will have to be started early to he of any
use, the babies are to be given VZV antiserum and
chem otherapy im m ediately after birth.
d ia g n o s is is u su ally
clin ic a l. M u lti nucleated giant cells and type A
intranuclear inclusion bodies may be seen in smears
prepared by scraping the base o f the early vesicles
(Tzanck smears) arid stained with toluidinr blue.
C ie m sa or Papanicolou stain, Electron microscopy
o f the vesicle fin id may demonstrate the vim* with
typ ictl heepet morphology. V iru s isolation tail be
attempted from the buccal or cutaneous lesions in
the early stages by inoculating hum an am nio n,
hum an fibrobhsr, I le L a or Veto cells. I'he virus
antigen cart he detected in scrapings Jrom skin
lesions by tmniLinoduorescenLe, and in vesicle fluid
by co u n te rim m u n o d c c tro p b o re s is wirh zoster
in iiminc serum. E L I S A and P C R techniques are
also in use.
A Live varicella
vaccine was developed, hv Takabasbi in Japan in
1974 hv attenuating a strain of varicella virus (O ka
strain, so named after the patient) by serial passage
in [issue culture. G iv e n subcutaneously, it induced
good antifnidy response, hut it was very Labile and
had to be stored frozen, A modi lied lyophUised
form o f the vaccine is now available, w hich can be
stored between 2 * C and b 'C It is recommended
for children 1 - 1 2 years old j s a single subcutaneous
dose, and for those older as 2 doses 6 - 1 0 weeks
apart. It is safe am! effective. O ccasionally children
may develop a tew vesicles w hich resolve quickly.
It is not considered safe in pregnancy. Varicella
zoster im m u n o g lo b u lin ( V Z I G ) prepared from
patients convalescing from herpes zoster provides
passive protection in immunocompromised children
exposed to in feet i on. hul it- aVulabiliTy is lim ited.
It if not useful in treatment.
S p e c ific treatm e n t if in d ic a te d m a in ly in
im niunndeficient and elderly subjects and those
w ith com plications such as varicella pneum onia,
encephalitis io d riissemirated zoster. Acyclovir and
fa m c ic lo v ir ir e effe ctive. C o r t ic o s t e r o id s arc
contra] ridiculed in varied I;las [lies' enhance the rink
o f pneum onia and disseminated disease.
T h e name is derived tnum H e /jid n , m eaning to
creep; zoster, meaning girdle.
While varicella is tvpically a disease of
childbfWKi, herpes zoster is one of old age, being
comm on after the age o f fifrv years. 'Lhe disease
may, however, occur ar any age and zoster has been
reported very rarelv even in the newborn.
H erpes zoster usually occurs in persons who
had c h ic k c rp o s several years earlier. T h e virus
rem aining larcnt in rhe sensory ganglia, may leak
out it times but is usually held in check hv the
residual immunity. Years after the initial infection,
when the im m unity has waned, the virus may be
reactivated, in ti triggered hy some precipitating
stim ulus, travel along rbo sensory nerve to produce
zoster lesions on rhe area o f the skin or mucosa
supplied bv it. T h is reactivation is associated with
inflam m ation o f the nerve, w hich accounts for the
neuritis' pain that often precedes the skin lesions.
T h e rash is typically unilateral and confined to the
area supplied by a single sensory ganglion. T h e most
comm on sites arc the areas innervated by spinal
Cord segments D 3 to L 2 and the trigeminal nerve,
particularly, its ophthalm ic branch. T h e Lesions arc
identical in nature to varicella lesio n ^ MCCpt for
their lim ned distribution. T h e rash heals in about
two weeks. Pain and paresthesia at the affected area
m ay p ersisr for w eeks o r m o n th s O th e r
com plications are lower motor neuron paralysis
which sometimes ensues^ m eningoencephalitis and
generalised zoster where the lesions arc scattered
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432 * TexibMk of Wici'Dtiioi-ogy
Prim ary infections in older children and adults
arc usually asymptomatic. However, a heterophile,
antibody negative, infectious m ontm udeosis may
be see r. I 'll is is more comm on fellow! ng transfer ion
o f C M V in fe c te d b lo o d ( p o s t -t r a n s f u s io n
m ononucleosi -).
In the im m unocom prom ised host, C M V can
cause Severe and even fatal infections. 'Phis occurs
in tra n s p la n t re< ip ie n ts , c a n c e r p a tie n ts on
chemotherapy, and m olt particularly in the H I V
infected. C M V is an important pathogen in A I D S -
In A I D S patients, the already weakened im m une
response is further dam aged by the nonspecific
C M 1 - in h ib it in g effect o f C M V . O n e o f the
glycoproteins cm the surface o f C M V acts as a
receptor for the f c portion o f im m un o g lo b ulin
molecules. T h is leads to m asking o f the virus by
attachment of irrelevant imm unoglohulin molecules,
preventing access to specific a n ti- C M V antibody.
L a b o r a to r y d iag n o sis; D L.Lgno--is m ay be
established by recovery o f the virus from the urine,
saliva or other body fluids by inoculating hum an
fibroblast cultures. A sim pler but less reliable
technique is the demonstration o f cytomegalic cells
in the centrifuged deposits from urine or s^ liv j.
D e m o n s [ration o f antib o dy is useful in rhe
d ia g n o s is o f p rim a ry in fe c tio n but not in
reactivation. Serological techniqties in use include
C F j I H A , I F and E L I S A , A ntibo dy detection may
be necessary for screening blood or organ donors.
B p id c m io L o CM V sp re a d s slo w ly an d
probably requires close contact lor transmission. It
may spread through salivary or other secretions or
by spm al contact. A special method o f transmission
U by blood transfuse m or organ transplants. T h e
virus has been detected in saliva, urine, cervical
secretions, semen, blood and m ilk. C o n g e n itally
infected infants have viruria for upto 4 -5 years. T h e y
arc highly infectious in early infancy. A bo ut one
per cent o f neonates in the U & A are infected w ith
C M V . I n the developing countries, the rate may be
m u ch higher. U p to 3 0 per cent o f adults show
C M V antibodies, indicating the high prevalence
*
o f infection. O n ce infected, the person carries the
vim s for life.
I J r e v e n t io n jicid t r e a t m e n t ! P re v e n tio n is
indicated o n ly in high risk cases such as organ
tran sp lan ts, i m m u n o d d le it nt p erso ns and in
premature infants. Screening o f blood and organ
d o nors and a d m in is t r a t io n of CM V
im m u n o g lo b u lin s have been e m p lo y e d in
prevention. Acyclovir is useful in pnopbykris hut
not in treatment. G a n ciclo v ir and foscarnet have
been [omul effective and are used for the treatment
of C M V disease in A I D S patents.
N o vaccine is available. Experim entally, live
attenuated vaccines (Tow ne 1 2 5 a n d A D 16 9
strains) and a purified C M V polypeptide vaccine
have been found to be im m u n o g e n ic but not
effective iti protecting im m unodefLcienr subjects
from C M V infcction.
EP5TEIN-BARR VIRUS
In 19 5 3 , Burkitt described an unusual lym phom a
am ong ch ild re n in certain parts o f A fric a and
suggested on ep id em io lo g ical grounds that the
rumour may be caused by a m osquito borne vims.
T h i * led to several attempts at i solat i ng viruses from
such tum o u rs. A num ber o f different viruses,
apparently 'passenger viruses', were isolated from
cultured lym phom a cells. O n e virus observed in
the cultured lym phom a cells by Epstein, Barr and
A chuitg in 196 4 was a new type o f herpesvirus,
nam ed the E B virus, specifically affecting cells o f
the B lymphocyte lineage. O n ly hum an and some
subhuman primate B cells have receptors [ C D 2 1
molecules) for the vim s, E1S V infected fi cells arc
tran sfo rm e d so th at th e y becom e ca p a b le o f
continuous growth in vitro.
E p id e m io lo g y : T h e E p s t e in -B a r r ( E B ) viru s
is ubiquitous in all hum an populations. A s w ith
other herpesviruses, infection w ith the E B vim s
leads to latency periodic reactivation and lifelong
persistence. T h e E B virus antibodies are present in
about 95 per cent o f adults. In the overcrowded
developing world, the E B vim s infection occurs in
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in f a n c y an d c h ild h o o d , w hen it is u s u a lly
asym ptom atic. In the affluent countries, prim ary
infection is often delayed till adolescence and early
a d u lt h o o d , w h e n it m a y le a d to in fe c tio u s
m ononucleosis.
T h e source o f infection is usually the saliva o f
in fe c te d p e rso n s w ho sh ed th e v iru s in
oropharyngeal seeretiuns for m onths follow ing
primary' infection and interm ittently thereafter
The EB virus is not h ig h ly contagious and
d ro p le ts an d ae ro so ls are nor e f f ic ie n t In
trams m itring infection. Intimate oral contact, an in
hissing, appears to he the predom inant mode o f
t r a n s m is s io n . 'I'llis a c c o u n ts for In fe c tio u s
monctfiuclectiis being called the 'kissing disease’.
In fe c tio n m ay a ls o fo llo w b lo o d or m arro w
transfusion but these arc rate events.
EcK virus infection may lead to the follow ing
clinical conditions:
1 . Infectious mononucleosis.
2. E B V associated malignancies:
a. BurkLtt's lym phom a.
h. Lym phom as in immunintefLcicnt persons such
as A I D S patients and transplant recipients.
c. Nasopharyngeal carcinoma in persons o f
C h in e se origin.
“H ie virus enters, the pharyngeal
epithelial cells through C R 2 {o r C D 2 1 ) receptors,
w hich are the same as for the C 3 d component o f
co m p le m e n t. It m ultiplies. loCallv. invades. the
bloodstream and infects B lym phocytes in w hich
two types o f changes arc produced. In most cases,
the vim s becomes latent inside the lym phocytes,
which become transformed or 'rmrrjorfuJjscid'su that
they become Capable o f indefinite growth in vitro.
T h e v are polyelonallv activated to produce many
kinds o f im m unoglobulins. T h e hederophile sheep
erythrocyte agglutinin seen characteristically in
infections m ononucleosis is an example o f such
polyclonal activation. A second type o f effect, shown
by a lew infected E eells is lytic infection, with cell
death and release o f mature prngency virions.
T h e m o n o n u cle o sis represents a po lyclonal
transformation, o f infected B lymphocytes. E B
vim s antigens are expressed on the surface o f
infected B cells. T h e atypical lym phocytes seen
in blood smears in infectious mononucleosis
are T ly m p h o c y te s u n d e r g o in g blase
transformation in response to such neoantigens.
Interm ittent reactivation o f the Latent E B virus
leads to clonal prnLilei.ition o f infected E cells. In
im m unocom petent subjects, this is kept in check
by activated"!" cells. In the im m unodcfieicnt, U cell
clo n e s m ay replicate u n c h e c k e d , re su ltin g in
lym phom as. H ypercndem ic malaria prevalent in
A frica is believed to be responsible tor the immune
im pairm ent in children w ith B u rk in ’s Lymphoma.
T h e frequency o f lym phom as seen in many types
o f im m unodeficiencies, most typically in A I D S ,
may have a sim ilar pathogenesis. N early h a lf the
lym p h o m as seen in im n u in o d c ftc ic n t subjects
contain E B virus D N A sequences.
G enetic and environmental factors are said to
be important in tile nasopharyngeal carcinom a seen
in m en lit C h in e s e o rig in . E B v iru s D N A is
regularly found in the tum our cells. These patients
have high levels o f E B virus antibodies. G enetic
In flu e n c e is best illu s tra te d in the X - l i n k e d
lym phciproliferative ( X L P or D u n can ) syndrome
associated with extreme susceptibility to E B virus
infection.
T h is is an acute self-lim ited illness usually seen in
no nim m iune y o u n g adult* fo llo w in g p rim a ry
infection with the E B virus. T h e incubation period
is 4 - S weeks. T h e disease is characterised bv fever,j
sore throat, Lympludettopathy and the presence o f
abnormal lymphocytes in peripheral blood smears.
A m ild transient rash may be present. Som e patients
treated w ith a m p ic illin m ay d e ve lo p a
m a cu lo p a p u la r rash due to im m u n e com plex
r t a o io n to the drug, T h e re is often associated
h e p a titis w h ic h is u s u a lly s u b c lln ic a l an d
demonstrable only by Liver function testis. A number
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Hidden page
 < Herpesviruses f 4 es

H um an H e r p e j V i r u s T > p e g b ,7 ,N H H V 7 also appears to be w idely distributed and

A herpesvirus, first iosolited in l9flb from the transmitted through saliva- Jt shares with H I V the

p e rip h e ra l blood o f p a tie n t? w ith lympho- same C D 4 receptor o n T cells and could therefore
■ contribute to a hirther depletion o f C D 4 1 Cells in
proHreranve disease, was called the hum an B -
lytnphotropic vims, It baa been renamed I I I I V - 6 , H I V infected persons. It has been said to cause

T h i - i s ubi; |ui ros and spreads apparent^ chm ^ogh wdb^ some cases o f exanthem subitum .
in early infancy. Tw o variants recognised, A and B, In 19 9 4 , D N A sequences presumed to represent
Variant B is the cause o f m i Id but common childhood a new herpesvirus were identified from tissues o f
ilinrsb ' cxaittheiti subitum’ (foScolri infantum uf visth Kaposi's sarcoma from A I D S patients. T h is has
disease: 1. In older age groups^ it has lvcen associated been named H H V S. T h is has subsequently been
with infcctinot mononucleosis syndrome, focal identified also in Kaposi's sarcoma in persons not
encephalic ^ and, iti the i-nm u node fie hint, w ith mfecced w ith H I V . h has been therefore referred
pneumonia and disseminated disease. to som e lim e s as Kaposi *f sa rco m a -assoc ia ted
H H V 7 was isolated in 19 9 0 from peripheral h e rp e s v iru s (K S H V ), but an e t io lo g ic a l
C D 4 cells o f a healthy person- Like H H V 6, relationship Is yet to be proved.

F u rtb a r HlihJ liil;

Curay I, and P Spear 19S6. Iti^tcrLotus with Htip« Simple* viruses. New Etig-J Med 3 J4:6W>, 74V.
Hu M. 1991. CjTQmqpaJoHnisej: Bk>Iogyrand Infection, 2 u edn. New Yirlc Plenum. Press.
Jaffc riW and PE Felleir 1999. Human herpesvirus 9 and Kaposi's sarcoma. New Engl J Med 24:140.
Kaplan JE. 1969. Herpesvirus simiae infection in monkry handlers. J Infect Dis. J571090.
Khatuna R. eial. 1995. Immune regulation in Epstein-Barr virus disease. Alicrobiol Rev 59:387.
LevyJA 199*. Three new human herpesviruses. Lancet349:558.
Lusao P and RC Gallo 1994. Human herpes virus 6 in AIDS. Lancer 343:5 55.
Onoraio 1M cr aL 1985. E|UilcmioJogy of cytomegalovim- infect .'tn. fiev /nterf D.<- 7;479.
Rickman A B et al. t995- TTlC Ep*tdn-B H T virus as a in' m l I n f virus-host ink r.n tiHnrta JJ. ' M e d B ull 41-.75.
R pw ky A H eC a l- 1990. Rapid detectiim itf H e rp a !-implcs virus DIVA. Jjnetfr 335.
Sirauis M-. li rl. 1993. Epsrcin-Bair virus infections. Ann Ini Med 1 19:45.
WtllerTH. 19E1. Varicella and Herpes-Zoster: changing concepts. New E ngJ Med. 3tf9l3&2, 1434.

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 Adenoviruses

A d e n o v iru s e s are a }»roup o f m e d iu m s iz e d , Adenoviruses appear to have a spare capacity

none nw Loped, double stranded D N A vi ruses that to carry D N A insert u p to 7 kb an d arc being
share a com m on com plem ent fining antigen. T h e v investigated as potential vectors i:; gene thcrapv.
infect human-s, anim als and birds, showing strict A d en o viru ses arc 7 0 - 7 5 nm in
host specificity, size, T h e y have ,t characteristic m orphology. T h e
In 1^ 5 3 , Rowe and associates grew surgically capsid is composed o f 2 52 capsomers arranged as
rem oved hum an adenoid tissue in jiLusma clot an icosahedron with 20 IriAtUpdar facets and 1 2
cultures and noticed that the epithelial outgrowths vertices. O f the 2 5 2 cap so m crs, 240 have six
underwent spontaneous degeneration resembling neighbours and arc called hexon-. while the 1 2
viral cytopatbic change. T h is was neutralised hv Lupsomeis at the vertices have five neighbours and
h u m a n sera. A viral agent was sh o w n lo he are called pentons. K w h perron unir consists o f a
responsible for this degeneration. T ills was the penton base anchored in rhe capsid and a projection
prototype o f the group o f viruses subsequently or fibre consisting o f a rod Like portion with a knob
d e s ig n a te d a d e n o v iru s e s b ecause th e y were attached at the distil end. Tims, rln- vir]on has the
originally isolated from the adenoids. I liUcman in appearance o f a space vehicle.
1 9 5 4 isolated a related v iru s from tile throat
washings o f m ilitary recruits with acute respiratory
illness.
O ver 5 0 serotypes o f adenoviruses have been
isolated from hum an sources. M o st o f the recenr
serotypes were recovered from A I D S patients.
A d e n o v iru s infectio ns are com m on w orldw ide
m o s tly in c h ild r e n . M any in fe c tio n s are
asym ptom atic, [’lie virus may persist in the host
for many months- Adenoviruses cause infections o f
the respiratory tract and eyes, and less often o f rhe
intestine ami urinary tract.
In 1 962 H ucb n er reported that adenovirus types
1 2 and 1 8 produced sarcoma when inoculated into
baby ham sters. T h is led to the intense studv o f
adenoviruses at the gene tic and molecular levels.
H o w e v e r, there is nn evidence a t i l l re la tin g
adenoviruses to natural m alignancy in hum ans or
anim als.

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A 12. IS , 3 1 Rat
B 3 ,7 , 1 1 ,1 4 . 1 6 ,2 1 ,3 4 ,3 5 Monkey
i: 1 ,2 , 5 , 6 Rat
i> 3 - 1 0 , 1 3 , 15, 17. 19*20*
2 2 -3 0 , 32, 33, 3*A$9,
4 2 -4 7
E 4 Rat
F ■ W, 41 Rar
Norei + denotes complete and t |■l±rTL;lI henugglLLEinaTion
A dncm nis-es are relatively stable,
rem aining viable for about a week at 3 7 aC , T h e y
arc readily inactivated at 50 ° C . T h e y resist ether
and bile salts.
A d e n o v iru se s are
host specific and so laboratory anim als arc not
susceptible to adenoviruses in fe ctin g h um an s.
H u m a n adenoviruses grow only in tissue cultures
o f human origin, such as human em bryonic kidney;
H e L a or H E P - 2 . Cvto path ll changes may take
several days to develop and consist o f cell rounding
and aggregation into grape like clusters. Intranuclear
inclusions m ay be seen in stained preparations.
T h e f a m ily A d e n o v ir id ie
c o n ta in s two g e n e ra 1 M ae la d en OVjrua, the
adenovirus o f m am m als and Aw adenow m s, that o f
birds. In addition co at least 47 serotypes o f human
origin, m astadenoviruses include sim ian, bovine,
eqtiinc, ovine, canine, m urine, porcine and cetacean
serotypes. A vi adenoviruses have been isolated from
fow ls, gelc and tu rkeys. T h e y infect o n ly the
h o m o lo g o u s s p e cie s, w ith th e e x c e p tio n o f
oncogenic hum an adenoviruses (for example types,
1 2 , 18 , 3 1 ) that cause sarcomas w hen injected into
newborn hamsters.
A ll mammalian adenoviruses share 2 common
complement fixing an Ligeti. T h e group antigens are
present m ainly on hexons and cart be detected by
t H igh *
+ W eak +
i Nil
■+ Nil
± Nil
l NK
N K = not known
irrui no flu o re sce n ce or E I T S A . T y p e -s p e c if ic
antigens arc located on pentonsand fibres. Serotypes
are identified by the neutralisation rest. H um an
adenoviruses arc classified into six groups (also called
subgroups or subgenera) based on properties such as
hemagglutination, fibre length, D N A fragm ent
analysis and orwogenic potential (Tahle 53,1)-
A denovi ruses cause infections o f
the respiratory tractf eye, bladder and intestine.
M ore than one type o f virus may pnsduce the ram f
clinical syndrome and one type o f vim s may cause
c lin ic a lly different diseases (T a b ic 5 3 -2 ) . T h e
following syndromes have been recognised:
Adenoviruses are the m ajor cause
o f non b a c te ria l p h a r y n g it is an d t o n s illitis *
presenting as febrile comm on cold. Types 1 - 7 are
com m only responsible.
A d e n o v iru s types 3 an d 7 ir e
associated with pneum onia in adults resembling
prim ary atypical pneumonia. In infants- and young
children types 7 may lead to more serious and even
fatal pneumonia,
T h is
occurs usually outbreaks in m ilitary recruits.
Serotypes 4, 7 and 2 1 are the agents com m only
isolated,
T h ia
syndrome o f febrile pharyngitis and conjunctivitis ■
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■j ; m esenteric lym ph nodes in eases o f mesenteric
, adenitis and intussusception in children.
seen in civilian population is usually associated with
serotypes X 7 and 14 .
■ ’
T h is ji$ a serious condition w hich may appear as
an epidemics* usually caused by type 8 and less often
by types 1 9 and 37.
T h is is a
nonpuculert inflam nnitian nt the conjunctiva w ith
enlargement o f the submucous lym phoid follicles
and o f the preaurivular Eymph nodes. Types 5, 4
and 1 1 are com m only responsible. Adenoviral and
chlamydial, conjunctivitis are clin ica lly sim ilar.
A d e n flv in iK S can often be iRotated
from feces but their relation to intestinal direarc
has not been conclusively established. How ever.
r* P
Respiratory disease in children
Sort throat, febrile cold, pneumnnia
A R D ill military n c n k l
Follicular (swimming pool) conjunctivitis
Epidemic keratucunjunttiviii- (shipyard eye)
Diarrhea
som e fa stid io u s a d e n o v iru s e s, w h ic h van he
dem onstrated ab u n d a n tly in feces by electron
m icroscopy but fail 10 grow in conventional tissue
vultures, can cause diarrheal disease- in children (for
example types 40. 4 1), 'J'hey have beer designated
as enteric type adenoviruses. Special techniques of
tissue culture (use o f trypsiruHcd m onkey kidney
cells or transformed hum an em bryonic kidney cells)
have been developed lor their cultivation. T h e y tan
also be identified by stool E L I S A .
A c u te hem orrh agic cystitis in ch ild re n and
generalised exanthem arc two other syndrom es
w hich have been reported. Adenoviruses types 1 1
and 2 1 are responsible lor the former.
A d e n o v ir u s e s h ave been is o la te d from
:* . .ji". D ia g n o s is c a n he
established by isolation o f the vim* from the throat,
CSX, urine ot feces. T h e materials a it inoculated in
tissue cultures. Preliminary identification is possible
by noting checytopathic effects and by com plem ent
fix a tio n tests w ith ad e n o v iru s a n tise ru m . B y
hemagglutination with rat and monlcev erythrucytes.
the isolate can be clashified into subgroups. Typ in g
in done by neutralisation tests,
F o r s e ro lo g ic a l d ia g n o s is , rise in ritre of
antibodies should be demonstrated in paired sera,
E x a m in a t io n o f a sin g le sam ple o f se ru m is
in c o n c lu s iv e as a d e n o v iru s a n tib o d ie s are so
com m on jn tbc population,
E le c t r o n m ic ro s c o p y fo r fe ca l v iru s an d
imm unofluorescence lo r viral antigen detection in
'*
1 ,2 .5 ,6
3 .4 , 7 ,14 , 31
4 * 7 ,2 1
V
0 .19 . 3?
40. 41
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 4 Ademvkws * 469

nasopharyngeal and ocular infections are useful. independently as they lack enough D N A . T h e y can
iV r ip h y la x ie c S p e c ific prevention required m ultiply only in cells sim ultaneously infected w ith
o n ly fo r rhc c o n tro l o f o u tb re a k s in closed, a d e n o v iru se s an d are c a lle d a d e n o -a sso cia te d
com m unities, as in m ilitary recruits. K illed and live viruses { A A V ) or adcnosatellitc virases. T h e y have
vaccines have been used in them for prevention o f been cla rifie d as the genus depencforinK {referring
A R D , w ith some success. Mo vaccine is available to their dependence on adenoviruses) under the
for general use. fa m ily P arv u v irid o e . T h e y can be detected by
election m icroscopy and com plem ent fixation or
ADEN O -ASSO CIATED VIRUSES (AAV) immunofluorescence w ith specific anti-era. Types
E lectro n m icroscopy o f adenovirus preparations 1. 2 and 3 are o f hum an origin and cause natural
have revealed small icnsahedral viral particles, 20 ~ infection, while type 4 is o f sim ian origin. T h e ir
25 nm in diameter. T h e y are unable ro replicate pathogenic role is uncertain.

Further Reading
li . L i r : ; S ( ? 1 9 9 0 . A d e r K h v in ic e x . I n ( j . M . i : : . I l, I r t j l . J V i ■n .m d P r a t r i i s c n f j n f i i ! hy.nr I .il1 i -il n . I V f w V > r fc W i l e y .
LX- K tr iL 19 li Fi/i'.LiH'iuii JciiiWHnYii+tJ iVHliti luiivnn ml jnl , li-il. J \ h ii l ijm J 1 I :J IV
Ginberj ] IS (ed). 1994. The AdtnoH'jrrarj. New York: PLccium.
ECcenly-ide RA l-i jl. 19B3. Kcramconjum rivicis isso.ljKtt with adenm :-ifus typ; 37, J /nfccf D i- 147:191,
Kemp M C er aJ. 19B3.The changing etiology o f epidemic keraroeonjuncriviTLi. J in fo * D i.t 14S:24.
fin C - 1991. ,4depr:v:r.i«5, ]n TerffrojJt (jfH vm w v V 'm ihgy, 2' «ln. BcErheiMurby Year Book*.

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 Picornaviruses
T h e fam ily Picom aviridae comprises a large number
o f very small (pew, m eaning small) R N A viruses.
T h e y art- noneiivelopcd viruses, ^ 7—3D nm in size,
resistant to ether and other lip id solvents. Tw o
groups o f picoroiviruses are o f m edical importance,
the enferoi'inrjej; that parasitise the enteric tract
and the rhijw vrnrw s th:u infect the n final mucosa.
T w o o th e r p ic o rn a v iru a g enera o f v e te rin a ry
importance are aphthoviruses causing the foot and
mouth disease o f cattle, and cardloviruscs o f mice,
including the eiicephalom yocarditis virus.
E n te ro v iru se s o f m edical im p o rta n ce in clu d e :
l\> lia v in is types 1 - 3 , Coxsackievirus A types 1 -2 4 ,
C a m c k ie r in u 13 types 1 - b , Echovirus types 1 - 3 4
and Enterovirus types 6 K -7 1 .
Paralytic dis«tse o f children (infantile paiafysis)
has been recognised from very early times. I lowever,
it was o n ly tow ards the end o f the nineteenth
century that poliomyelitis (polim = grey; myelitis
= i nflanunarion rif rhe spinal coed) was eharactensed
as a separate c lin ica l entity capable o f cau sin g
in fe c tio n s in w h ic h p a r a ly tic case s are tar
o u tn u m b e re d b y sile n t in apparent in fe ctio n s,
L in d s t e in e r an d Popper (19 0 9 ) reported
experimental tran sm i^ io n o f the disease tn monkeys
by ino culatio n o f sp in al cord and t’c cal extract
filtrates from fatal cases o f p o lio m y e litis. T h e
experimental study o f the disease was restricted as
m o n k e y s w ere the o n ly la b o ra to ry an im a ts
s u s c e p tib le to th e v iru s . A r m s t r o n g ( 1 9 3 9 )
succeeded in adapting a poliovirus (type 2 L an sin g
strain) to cotton rats and mice but few strains could
be so adapted. Progress was also inhibited by die
dogma then held that polioviruses were strictly
ncurotropic, m ultiplying only in the neural [issues.
T h e demonstration by E r d c n , V\fcllcr and Robbins
(1949) that polioviruses could grow in cultures o f
ncm-rteurj] cells from hum an embryos, producing
cytopathk effects, was a major breakthrough. T h e
Nobel Prize was awarded to them in recognition
o f the sem inal importance o f this discovery in the
development o f virology as a whole.
A new type o f virus was isolated by D a lld o rf
and Sicldcs (1948) from the feces o f children with
p a r a ly tic p o lio m y e lit is , fro m w h o m type 1
poliovirus- was also isolated- T h e virus caused
paralysis on inoculation into suckling mice. T h is
was called the coxsackk virus, as the patients came
from tilt village o fC u a sa ck ie in N ew York. M any
sim ilar viruses have since been isolated from the
feces and throats o f patients with different diseases
as well as from healthy individuals. T h e y have been
designated asttwsackic viruses, classified into groups
A and U based on the p a th o lo g ic a l ch a n g e s
produced in suckling mice.
T h e introduction o f tissue culture techniques
in diagnostic virology led to the isolation o f several
cytopathogenk viruses from the feces o f sick as
well as- healthy persons. T h c v were called orphan
viruses as they could not be associated w ith any
particular clinical disease. T h e y came to be known
bv the descriptive Term 'enteric evtopathogenic
hum an orphan ( E L ’ I i O ) viruses', Several orphan
viruses have also been isolated from anim al frees.
The classification of enteroviruses as
c o x s a c k ie v iru s e s and e c b o v iru s e s was not
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4n * T&tjttooofc of MIcrobMogy >
H i e v im s grows readily in tissue cultures o f
primate origin, Prim ary monkey kidney cultures are
used fur diagnostic cultures and vaccine production.
T h e infected cells round up and become icfractilc
and pyknoticr Eo sino philic intranuclear inclusion
bodies may be demonstrated i n sta i ned prepsiat ions.
W e ll-f o r m e d p la q u e s d e ve lo p in in fe cte d
monolayers w ith agar overlay.
P a th o tfe n c ft!^ T h e virus is transm itted by the
fecal-o ral route through ingestion. Inhalation or
entiy through conjunctiva o f droplets ofrespiratory
secretions may also be possible modes o f entiy in
close contacts o f patients in the early stage o f the
d ise a se . T h e v iru s mu I n p lica in it ia lly in the
ep ith e lia l cells o f the alimentary' canal and the
lym phatic tissues* from the tonsils to the Feyer’s
patches. It then spreads to the regional lym ph nodes
and enters the blood stream {m in o r or prim ary
v jV fjn ja ). A f t e r fu rth e r m u lt ip lic a t io n In the
reticuloendothelial system , the virus enters the
bloodstream a # i i n { m ajoror scctwidaiT virernjaJ and
i s carried to the sp i nal cord and brai n. D i reel neural
transmission to the central nervous system may also
o c c u r u n d e r s p e c ia l c irc u m sta n c e s* as in
poliom yelitis following tonsillectomy.
I n th e c e n tra l n e rv o u s sy ste m , th e v iru s
m ultiplies selectively in the neurons and destroys
them. T h e earliest change is the degeneration o f
MiS}.1 bodies (c b rn m a tn lv rii), N u c le a r clicr.^es
follow. W h e n degeneration becomes irreversible,
the n e cro tic cell lyses o r is p h ag o cytn se d by
leucocytes or macrophages. Lesions are mostly in
the anterior horns o f the spinal cord* causing flaccid
paralysis, but the posterior horns and intermediate
co lu m n s m ay also be involved to som e extent.
Pathological changes are usually more extensive
than the distribution o f paralysis. In some cases,
e n c e p h a lit is o ccu rs p r im a r ily in v o lv in g the
b ra in ste m but e x te n d in g uptn the m o to r and
premotor areas o f the cerebral cortex.
C l i n i c a l f e a t u r e : F o llo w in g expo sure to
poliovirus, 9 0 -9 5 per cent o f susceptible indivi duals
develop only ioapparenf infection, w hich causes
seroennvenion alone. It is only in 5—1 0 per cent
th at an y so rt o f c lin ic a l illn e s s re su lts- T h e
incubation period Is about 1 0 days on the average
but may Tange from four days to four weeks. T h e
earliest man I testations are assoc i-Lted w ith the phase
o f prim ary virem ia and consist o f fever, headache,
sore throat and malaise lasting 1 - 5 days. T k iv is
called the m ino r illness and in m any cases may be
the only manifestation (abortive paliumrziitis). I f
the infection progresses, the minor illness is followed
H days later by the m ajor illness. T h e fever comes
o r aga i n [birh as ic fever), along w ith headache, stiff
neck and other features o f m eningitis- T h is marks
the stage o f viral invasion o f the central nervous
system. Sometimes the disease does not progress
beyond this stage o f aseptic m eningitis (nonparsJyric:
poliomyelitis). I n those proceeding to paralytic
polinm yeliris, flaccid paralysis develops. Paralysis
is local in distribution initially but spreads over the
next 3 -4 days. D epending « u the distribution o f
paralysis, cases are classified as spinal, bulbar or
bulbospinal. M ortality ranges from 5 - 1 0 per cent
and is m ainly due to respiratory failure. Recovery
o f the paralysed muscles takes place in the next 4
8 weeks and is usually complete alter six m onths,
leaving behind varying degrees o f residual paralysis.
L a b o r a t o r y ilin g n o a is t V ir u s is o la t io n in
tissue cu ltu re is the best m e th o d for sp e cific
diagnosis. M any specimens can be used, including
blood, C S F , throat swab and feces. V im s can be
isolated from blood during the phase o f prim ary
viremia, 3 - 5 days after infection, before neutralising
antibodies appear. B u t th is ls o f little practical
importance. U n lik e other enteroviruses, poliovirus
can seldom be isolated from the C S F but can be
o b ta in e d fro m th e s p in a l co rd an d b r a in ,
postm ortem . T h e virus can be isolated from the
th ro at iu the early stage o f the disease. V iru s
isolation from feces is usually possible from over
SO per cent, o f patients in the first week, 5 0 per
cent- till 3 weeks and 2$ per cent, till six weeks. A s
fecal excretion may be intermiitent* best results are
obtained by testing fecal samples collected on two
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separate days, as early in the illness as possible.
F r u l u i i g d fecal excretio n m ay be seen in the
i :ii:i i lj 11 otl-e; Jif i t . but perm anent carriers do nut
occur.
After appropriate processing to destroy bacte.i la
[centrifugation, treatment with ether, addition o f
antibiotics), specimens are inoculated into tissue
culture. Prim ary m onkey kidney cells are usually
em ployed, though any other hum an of simian cell
culture m ay be used. T h e virus growth is indicated
by t y p ic a l c y to p a rh ic e ffe cts in 2 - 3 days.
Identification is made by neutralisation tests with
pooled and specific antisera. It m ust be remembered
that the mere r-olatian u f poliovirus frnm feces does-
not co n stitu te a d ia g n o sis o f p o lio m y e litis as
sym ptom less in fe ctio n s are so co m m on. V ir u s
isolation must be interpreted do ng w ith clin ical
and serological evidence.
Scrodiagnosii is less often employed. A n tib o d y
rise cun be d e m o n stra te d in p a ire d sera by
n e u tra lis a tio n nr co m p lem en t fix a tio n tents.
Antibodies appear soon after the onset o f paralysis
so that even the first sample o f serum may contain
appreciable am ounts o f antibody. N e u tra lis in g
antih<jdies appear c.iclv and pefsifl for life. In the
C F test, anti bod ics to the C .mti^cn appear first
and d isap p ear in a few m o n th s, w h ile a n t i-D
antibodies take some weeks to appear after infection
but last for five ycars.Thc C F test is useful to Identity
exposure to polio virus but not for type s p e c if ic
diagnosis.
I m m u n it y : Im m u n ity in p o lio m yelitis is type
specific- H u m o rs I im m unity provided by circulating
and secretorv antibody is responsible far protection
against poliom yelitis. Ig M antibody appear? w ithin
a week o f infection and lasts for about six months.
I g G antibody persists for life. Neutralising antibody
in blood generally protects against disease In' the
same serotype o f the virus, but may not prevent
infectio n o f intestinal ep ithelial cells and virus
s h e d d in g in feces. S e c re to ry I g A in the
gastrointestinal tract provides m ucosal im m u n ity
preventing intestinal infection and virus shedding.
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Breast m ilk containing Ig A antibody protects infants
from infection.Poliom yelitis rends to be more severe
and virus shedding m an; prolonged i:i those with
impaired hum oral imm une response.The virus also
induces cell mediated im m unity, but its importance
appears to be uncertain as persons w ith defective
cellular im m unity arc seen to respond norm ally to
poliovirus infection.
P r o p h y L a s r if t P a ssiv e im m u n is a tio n by the
adm inistration o f human gam m aglobulin is o f little
value.
Attem pts at active im m unisation w ith vaccines
date fro m 1 9 1 0 , Soon after the d isco v e ry of
poliovirus. The e a rly vaccines w ere cru d e
su sp en sio n s o f the sp in a l cord fro m in fected
monkeys, inactivated w ith form alin (Brodie and
Park) or ridmolealE (Kolm er). T h e y were not o n ly
ineffective but often even dangerous, leading to
vaccination poliom yelitis. Folio vaccines therefore
became unpopular. Brodie is believed to have taken
his own iife, distressed at the suffering caused by
his vaccine. It was o n ly after 19 4 9 , when tissue
culture was used for grow ing the virus and the
existence o f three antigenic types o f polioviruses
was recognised, that fresh developments in vaccine
preparation became possible. B y 1953, S alk had
developed a killed vaccine. A lm o st simultaneously,
K o p ro w skyC o x and Sabin independently developed
live attenuated vaccines.
S a lk 's k ille d p o lio v a c c in e is a f o r m a lin
in activ a te d p rep a ratio n o f the three types o f
poliovirus grown in monkey kidney tissue culture.
Standard virulent strains arc used. T h e three types
o f polioviruses ire grow n separately in m onkey
k h Iney cells. V ira l pools o f adequate ri tre are filtered
to remove cell debris and clum ps, and inactivated
w ith form alin (1:4 0 0 0 ) at 3 7 ° C for 1 2 - 1 5 days.
Stringent tests are carried out id ensure complete
inactivation and freedom from extraneous agents.
T h c three types are then pooled and after further
tests for safety and potency, issued for use.
A nationwide controlled Held trial conducted
in 19 5 4 in the U S A confirm ed rhe safety o f the
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4 9 fi * " t e x tb e o k tyf M u r o b io lo g y *
disadvantage hi the advanced cuuntnes. ] rbi.11's, iL
may even be beneficial and m ay help to extend the
vaccine coverage in countries where w ild vim s is
e n d e m ic . Id e a lly , ho w e ve r, it is d e sirab le to
vaccinate the whole com m unity ,u one time so that
natural dissemination is prevented. The strategy of
adm inistering the vaccine to ail children in a region
o n the same day {pulse ifimmiiitition) has been
found to be useful in the developing countries.
E r a d i o q l i o n o f p o l i o m y e l i t i s : By g lo b a l
im m unisation w ith O P V it was considered possible
to e ra d ic a te the d ise ase . T h e W o r ld H e a lt h
O rg a n isa tio n A sse m b ly in 19 fiS had proposed
global cradica rion o f pel iomycl'i ri s by the year 2000-
Poor progress in im m unisation in m any countries
has been a set back to this objective.
E p id e m io lo g y : Po lio m ye litis is an exclu sively
h u m a n disease and the o n ly source o f virus is
humans, the patient or m uch more cam m onlv the
symptomlcss carrier. Patents shed rhe virus in feces
for varying periods, about 5 0 per cent for three weeks
an d a s m a ll p ro p o rtio n far 5 - 4 m o n th s. N o
permanent earners occur, 1 lowcvcr, the virus may
persist in the environm ent (sewage) for upto six
m onths. V im s shed in throat secretions dui mg the
early part o f the di-ease may also be a source of
infection for the contacts o f patients.
Infection is, in general, asymptomatic. T h e ratio
o f subclm ical to clinical infections has been stated
as 10 0 or 10 0 0 to 1. T h e outcome of infection is
influenced by the virulence o f the infect:tig strain,
the dose o f infection and the age o f the indiv idual,
adults being more susceptible than children. T h e
follow ing factors m ay influence the incidence o f
p&Taly^!-: 1 ) Prcgnancv carries an increased risk o f
paralysis, perhaps due to the associated horm onal
changes. 2 } To nsillectom y during the incubation
period may predispose to bulbar poliomyelitis.
3) I rejections such as triple vaccine, especially aluiri-
co n tair.in g preparatio ns. m ay lead to paralysis
involving the inoculated lim b- T h e m echanism is
uncertain. T h e trauma m ay lead to virus entry into
local nerve fibres, or the segment o f spinal cord
corresponding to the site may be more susceptible
to viral damage due to reactive hyperem ia 4) Severe
muscular exertion or trauma dui mg the preparalymc
stage increases the ride o f paralysis.
P o lio v ir u s type 1 is re sp o n sib le fo r m o st
epidemics of paralytic poliomyelitis. Type 3 also
causes epidem ics to a lesser extent. Type 2 usually
causes inapparent I nfections in the western countries
but in India paiafyi iv-due to type 2 is quite common.
Immunity is type-specific but there is an significant
am ount o f cross protection between types 1 and 2 ,
between types 2 and 1 and little or none between
types 1 and 3.
COXSACKIEVIRUS
T h e prototype strain was isolated by D a lid o rf
and Sicldes (19 4 3 ) from the village o f Co xsackie
in N e w York. Several related viruses have been
i Kolated m ncc then from di fferent parts o f the world,
T h e characteristic feature o f this group is its ability
to infect suckling hut not adult mice, Based on the
pathological changes produced in suckling m ice,
coxsackieviruses are classified into two groups, A
and B.
P r o p e r t i e s td" t h e v i r u s : C o x s a c k ie v iru s e s
arc typical enteroviruses. Follow ing inoculation in
s u c k lin g m ice , g ro u p A v iru s e s p ro d u c e a
generalised myositis and flaccid paralysis leading
to death w ithin a week. G ro u p B viruses produce a
patchy focal myositis, spastic paralysis, necrosis o f
the brown fat and, often, pancreatitis, hepatitis,
myocarditis and encephalitis- B y neutralisation tests,
G ro u p A viruses are classified into 2 4 types and
group B into six types. A ll types in group B share a
comm on com plem ent-fixing antigen- Co xsackic A
23 is the same as echo 9 and Coxsackie A 2 4 the
same as E C H O 34, Som e coxsackieviruses ( A 7,
20, 2 1 , 2 4 and B 1, 3, 5 T 6) agglutinate hum an or
m onkey erythmeytes-
H o * t m a g e a n d g r o w t h : It is necessary to
e m p lo y s u c k lin g m ice fo r the is o la t io n o f
coxsackieviruses. Inoculation is usually m ade by
intracerebral, subcutaneous and Lntraperitaneal
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 * Picomav ruses ►
mutes. A d u lt m.<.x a lt not susceptible. Su cklin g
hamsters can be iiifeited experimentally.
A|) coxsack'n: E v' ruses grow well in m onkey
kidney tissue culturet, while in group A , only types-
7 and 9 straw welt. G ro u p A 21 virus prows in
H e L a cells.
C l i n i c a l f e a t u r e s : C o x sa ck ie v iru se s produce
a vancty o f clinical syndromes in hum ans ranging
from trivial to fatal infections. T h e following types
have been recognised:
1. H erpangiua (vehicular ptiaiyngiris} is a common
c lin ic a l m in i testation o f coxsackle group A
in fe ctio n in c h ild r e n . Tt is a severe febrile
pharyngitis, w ith headache, vom iting and p;vn
l i i the abdomen. T h e characterlsiic lesions are
sin a il vesicles, on the fauces and po sterio r
pharyngeal w all, that break down to form ulcere,
2- Ascpt ic men iugiri-. m ay be caused by most group
A and all group Q v iruses. A maculopapular rash
may be present. T h e dL^ease m ay som elim es
o c c u r as e p id e m ic s. T y p e A 7 had caused
outbreaks o f paralytic disease in Russia, Scotland
and elsewhere, the virus for a time having been
erroneously referred to as Poliovirus type 4,
3. H a n d -F o o t-a n d -M o u th -D ise a se { H F M I l ) was
identified in I9 6 0 as an exanthematous fever
affectmg m ainly young children., characterised
by dusters [if papulxives ic llL.lt le> ions on the ski n
and oral mucosa. It occurs as sporadic cases and
as outbreaks, C o xsackic A - 1 6 , 9; B 1 - 3 were
Com m on causative agents in itially . It was a
h eni^n illness reso lvin g in 1 - 2 weeks. T h e
situation changed drastically in the 19 7 G rs w ith
e n te ro v iru s-7 1 b eco m in g a causative agent,
c a u s in g exten sive e p id e m ic s w irh se rio u s
c o m p liv s t i ons lik e a se p tic m c r in g i : is ,
e n c e p h a litis , fla c c id p a ra ly s is , p u lm o n a ry
hem orrage, w ith m any fatalities, p articu la rly
in East A sia fru m Tai wan to Sin^ppure. H F M D
in now an important emerging disease.
4. M in o r respiratory infections resembling com m on
cold may be caused by A 10 , 2 1 , 24 and B 3.
4. Epidemic pleurodynia or Bornholm disease (so
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497
called because i.t w as first described o n the
D a n is h island o f Bornholm ) is a febrile disease
w ith stitch-like pain in the chest and abdomen,
caused hy group B viruses. T h e di&casc m ay
occur sporadically or i ; epidemics.
6. Myocarditis and pericarditis In the newborn,
associated w ith high fatality may be caused hy
group B viruses. T h e disease m ay som etim es
occur in older children and adults also.
7 . J u v e n ile d ia b e te s has been c la im e d to be
associated w ith coxsackle B 4 infection but a
c a u s a l ro le for t h is v iru s has not been
cstablished-
8. O rch itis due to coxsackievirus has also been
reported-
9. Transplacental and neonatal transm ission has
been dem onstrated w ith coxsackle b viruses
resulting in a serious disseminated disease that
[] i ,lv include hepati; IB, meningoencephalit is and
adrenocort i cal involvement.
10. T y p e E viruses have been associated with the
condition called posrt iraf fatigue syndrome, hut
neither the condition nor the association has
been clearly defined.
L a b o r a t o r y d iii^ n o M S : V iru s Eo latio n from
th e Lesions o r fro m feces m ay be m ade by
inoculation into suckling mice. Iderl liication is by
studying the hiRtnpathnlogy in. infected mice and
by neutralisation tests. D u e to the existence o f
seve raJ a n tig e n ic typ e s, se ro d ia g n o sis is not
practicable.
Jvf .tle n LNirNji'h'L T. ike o th e r e n te ro v iru s e s ,
co x sack ie v iru se s in h a b it the alim e n ta ry ca n a l
prim arily and are spread hy the fecal—oral route-
Co xsackle B virus ephkm ics tend to occur every
2 —5 years. Y o u n g infants are m ost co m m o n ly
affected. Vaccination is not practicable as there are
several serotypes and im m unity is type specific.
ECHOVIRUSES
W h e n tissue cultures became routine procedures
i n diagnostic virology, several cytopathogenic viruses
came to be isolated from the feces uf sick as well ££
healthy individuals. I ’hcsc viruses were not pathogenic
fm lalxirJDtrfv am mals.They were neutralised bypoolcd
hum an gam m i g lo b u lin . A r they co u ld not be
associated with any particular clinical disease then,
they were called orphans. T h e y have been given rhe
descriptive designation ^enteric cvtopithugEnic human
orphan viruses' and arc generally known by the sigta
echoviruses1. Sim ilar 'orphan' viruses have also been
isolated from many animals.
E ch o v iru ses
rescmhlc O ther enteroviniHes in their properties. By
neutralisation tests, they have been classified into
■ 34 serotypes, Types 10 and 28 have been rem(JYed
from the group, the former becom ing a rcovirus
and the latter a rhinovirus.
Som e echoviruses (types 3, 6, 7, 1 1 , 1 2 t 13 , 19 .
2 0 , 2 1 , 24 , 2 9 , 31) and 3 3 ) agglutinate hum an
erythrocytes. H e m a g g lu tin a tio n is follow ed by
elution, rendering the cells inagglu finable by echo
ut coxsackieviruses but not by myxoviruses.
A l l e c h o n ju se s g ro w
w e ll in h u m a n an d h lm ian k id n e y c j ] 11 1 res
producing cvtopatliic effects. EcboviiuSeB infect only
hum an beings naturally I'hey arc not pathogenic
ro laboratory animals chough occasional strains may
produce paresis 0(1 inoculation into monkeys and
newborn mice.
T h o u g h echoviruses were
originally considered oqiltans thev have since been
shown ro produce a variety o f disease patterns. M ost
infections are asymptomatic. In general, the clinical
fe atu re s re se m b le those p ro d u ce d by
co x sack ie v iru se s, Fever w ith rash and aseptic
m e n in g itis , so m e tim e s as e p id e m ic s , can be
produced by several serotypes, predom inantly by
types 4 , 6 , 9 , 1 6 ,2 0 ,2 8 and 30. Echoviruses perhaps
co n stitu te the m ost co m m o n cause of useptic
m e n in g itis . E c h o v iru s e s have frequently been
isolated from respiratory distune in children (types
1, 1 1, 19 , 2 0 and 22) and gastroenteritis (type 18 ),
but rh clr etio lo gical role Iras not been proved.
O ccasio nal cases o f paralysis and hepatic necrosis
have also been reported.
F e c e s, throat swabs;
or C S F m ay he i noculated i nto monkey kidney tissue
cultures and virus growth detected by Cytopaihic
ch arg e s- T h e lu g e num ber tif serotypes makes
identification by neutralisation tests laborious. T h i i
may he sim plified by hemagglutination and the use
o f serum pools for n e u tralisatio n . S e ro lo g ical
d ia g n o sis is not p racticab le except in case o f
epidem ics where the causative serotype Has been
identified.
L ik e o th e r e n te ro v irtu e s ,
echovirascH inhabit the alim entary tract prim arily
and are spread hy the fecah-or.il route. Epidem ics
may occur, especially in summer. Vaccination has
not been attempted.
G t the new enterovirus types, 6 S - 7 1 , type 68 was
isolated from pharyngeal secretions o f children with
pneumonia and brunch] Li*. Type 69 is not associated
w ith any human disease. T y p e 7 0 cause h acute
h e m o rrh ag ic C o n ju n ctiv itis. E V - 7 1 , o rig in a lly
isolaced from cases o f m eningitis and encephalitis,
causes many other syndromes, including H F M D ,
A pandem ic o f acute hemorrhagic conjunctivitis,
apparently arising in W est A frica in 19 6 9 spread
widely involving several parts o f A frica , the M iddle
East, India, South East A sia, Japan, Eng land and
Europe. T h e incubation period for this virus is
about 24 hours and the sym ptom s are sudden
swelling, congestion* watering and pain in the eyes.
S u b co n ju n ctiv al hem orrhage is a characteristic
feature. There is transient corneal involvem ent but
re c o v e ry is u s u a lly c o m p le te in 3 - 7 days.
R a d ic u L o m y e lo p a th y h as been r epor t ed as a
com plicatio n from In d ia . Som etim es it leads to
paralysis resembling poliom yelitis.
T h e causative agent was identified as enterovirus
type 70. It grows o n ly on cultured hu m an ce lls
(hum an em bryonic kidney or H e L a ) on prim ary
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 Orthomyxovirus

The name Alyxovirus was used originally for a
group o f enveloped RNA viruses, characterised by
rhcir ability' ro adsorb onto mucoprotciri receptors
on erythrocytes, causing hemagglutination. The
name referred to the affinity nfthe viruses Co mucins
(from myxfl, meaning mucus). Included in this
group were influenza,, mumps, Newcastle disease
and parainfluenza viruses. The subsequent
recognition o f Important differences between
influenza viruses and the other viruses in this group
led to their being reclassified into two separate
. famihes ~ orrhomy^owfitJae consisting of the
iilfluenzu viruses and pam m yxoviridlc consisting
o f the Newcastle disease virus, mumpsvirus,
para influenza viruses, measles and respiratory
syncytial viruses. Table 55-1 lists the important
differences between orthom yxovirus and
paramyxovirus.
Influenza is an acute infectious disease of the
respiratory tract which occurs in sporadic, epidemic
and pandemic forms. The name influenza is said
to have been given by Italians during the epidemic
of 174.1, which they ascribed to the malevolent
influence of the heavenly bodies or of inclement
weather. The modern history' of the disease may' be
considered rodatc from the pandemic of 1 RH9-1 H90r
during which Pfeiffer isolated Haemophilus
influenzae and claimed that it was the causative
agent. The most severe pan demic occurred in 1.918 -
1919, when it was shown that Pfeiilerhs bacillus
was not the primary cause o f the disease, though it
might act as. a secondary invader. The isolation o f
the influenza virus in 1953 by Smith, Andrewcs
and Laldlaw was a milestone in the development
of medical virology. They reproduced the disease
in ferrets by intranasal inoculation with hacreria-
free filtrates of nasophatyngeal secretions from
patients. Burnt [ (1935) developed cluck embtyo
technique:*; for the propagation of the virus.
A nntable advance was the independent

5iT.fi t»f virion !:HI 120 mu 100 300 Ain

Shape Spherical; filaments in fresh isolates Pleomorphic
Genome Segmented; eight piece* of RNA Single linear
molecule of RNA
Diameter of nucleoeapsid 9 mu 1$ tim
5ite of synthesis of ribonucleoprotein Nttcltut Cytoplasm
Genetic leassortmenf Common Ahsem
UNA-dependent RNA synthesis Required for multiplication Nut required
Effect of Actinomvein L> Inhibits multiplication Does not inbibLt
Antigenic stability Variable Stable
Hemolysin Absent Present

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502 * Textbook o ' Microbiology
discovery by Hirst, and by McClelland and Hare
(1941) chit influenza viruses agglutinate fow]
erythrocytes. The property of hemagglut inatk m was
found to be a common feature of many other Anises,
Francis and M agi 11 (1 9 4 0 } independently
isolated a serotype o f influenza virus which was
antigenicalfy unrelated to the strains known till then.
This was designated influenza virus type E ■ to
distinguish it from the original serotype, which was
named type A. Taylor (1949) isolated the third
serotype of influenza vi ms, type C . The clasui ficat on
of influenza viruses liiLci the three serotypes, A, B
and C , is based on the antigenic nature nf the
'internal1 or n.bonucleoprorein and the matrix (M)
protein antigens-
Influenza occurs also in animals and birds in
nature. Indeed, the avian influenza virus was
demonstrated as early as in 1901, when Centanni
and Avonuzzi showed that fowl plague was a viral
disease. However, as fowl plague (avian influenza)
is a septicemia, so different dinically from human
influenza, the association between the two remained
unknown till 1955, when Schaefer demonstrated
that the (owl plague virus was anrigcnically related
to type A influenza virus. Shope (1931) isolated
the swine influenza virus- N ot only did the swine
disease resemble human influenza clinically but
there was also cpiLlcmiological association between
the two. It was widely held that the virus spread to
swine from man at the lime of the 1918 pandemic.
Influenza viruses have also been isolated from
horses., whales and sc.lIh.
Birds, particularly aquatic birds, appear to be
the primary reservoir of influenza viruses and
natural infection has been identified in several avian
species. In birds it is usually an asymptomatic
intestinal infection. The cloaca of healthy wild birds
is the best source for isolation of avian influenza
viruses. All isolates from nonhuman hosts belong
to type A- Influenza virus types B and t- appear to
be exclusively human viruses and natural infection
with them has not been identified in animals or
birds. Ordinarily, non human influenza viruses do
*
not cause hum an infection. But they may play an
important role in the emergence of pandemic
influenza.
In f lu e n z a V iruses
M o rp h o lo gy : The influenza virus is typically
spherical, with a diameter o f 8 0 - 1 2 0 nm but
plcomoiphi^m is common. Filamentous forms, upto
several micrometres in length and readily visible
under the dark ground microscope, are frequent in
freshly Isolated strains.
The virus core consists o f ribonucleoprotein in
h c’ical symmetry. T h e negative sense single
stranded RNA genome Is segmented and exists as
lijtIl; pieces. Also present is a viral RNA-dependenl
RNA polymerase which is essential for transcription
of the viral RNA in infected host cells. The
micleocapsid is surrounded by an envelope, which
has an inner membrane protein layer and an outer
lipid layer. The membrane protein in also known
an the matrix or 'M protein' com posed o f 2
components, M 1 and M 2. The protei 11 part o f the
envelope is virus coded hut the lipid layer is derived
from the modified host cell membrane, during the
process of replicat ion by budding. Projecting from
the envelope are two types of spikes (peplomers):
fttrjTiagigJ'iJlJJo.rr spikes which are tri;ingular in cross
section and the mushroom shaped ncLrramiJifdj.se
peplomers which are less numerous (Fig. 5S.1).
R esistan ce: The virus Is inactivated by heating
at 50 “C (or 30 minutes. It remains viable at 0 -4
for about a week. It can be preserved for years at
-7 0 °C or by freeze drying. The virus survives slow
drying and may remain viable on fumites such as
blankets for about two weeks. Ether, formaldehyde,
phenol, salts of heavy metals and many other
chemical disinfectants destroy injectivity, Iodine is
particularly effective.
Hcmagglutinatiug, enzymic and complement*
fixing activities of the vims are more stable than
infectivity.
H e u n a g jg ju tin io n : Hemagglu-.ination is an
important characteri -ri c of influenza vi ruses. When
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 * Orthmiyxcivkiis ■j
cspc-.'ij]ly m type B infection in <r>.ildren, which
may even present ,i. e an acute abdominal emergency.
The uncomplicated disease resolves within about
seven lI.lv-.
Thcmosr important complication i-pneumonia,
which is mainly due to bacterial supcrinfcction or,
rarely, caused by the virus itself. C ardiac
com plications,, such as c c n ^ i t i v e failure or
myocarditis and neurological involvement, such as
encephalitis, may occur rarely.
Influenza, particularly infection with type B, has
beer associated with Reye's syndrome. It especially
affects young children and is characterised by acute
degenera iive changes iilthe bru 11i, liver and kidneys.
Type B infec:ions may som etimes cause
gastrointestinal symptoms (gastric flu).
L a b o r a to r y diagnosia: l . £>titwrtsmntQti o f
the nrws antigen- Rapid diagnosis of influenza may
he made by dem onstm ior of the virus anri^cr on
the surface o f the nasopharyngeal cells by
immunofluorescence. Detection o f the viral RNA
by reverse transcriptase polymerase chain reaction,
is extremely sensitive, but is o f limited availability,
2. Isolation o f the virus: Virus l-olaiion is obtained
readily fmm patients during the first two or throe
days of the illness hut less often in later stages.
Throat gargling* are collected using broth saline
or other suitable buffered salt solution. If the
specimen is not processed immediately, it should
be stored at 4 ^C,. or if the delay is long, at - 7 0 riC.
The specimen should be treated with antibiotics
to destroy bacteria. Isolation may be made in eggs
or in monkey kidney cell culture.
T he material is inoculated into the amniatic
cavity of 11 -1 3 day old eggs, urging at least six eggs
pet specimen. After incubation at 35 X ’ For three
days, the eggs are chilled and the amniotic and
allantoic Quids harvested separately. The Quids aie
tested for hentaggluri nation using guineipig and
fowl cells in parallel, at room temperature and at
4 *Q. Some strains of the influenza virus tvpe A
agglutinate only guineapig cells on initial isolation.
The type B virus agglutinates both cells, while type
5 07
C strains agglutinate only fowl cells at with amiseru
to types A, R .md C. Subtype identification is made
by hemagglutination inhibition test. Some of the
recent type A strains can be isolated by direct
allantoic inoculation of the clinical specimen into
9 -1 1 day old eggs. However, type B and C viruses
will be missed if only allantoic inoculation is used.
Inoculation into monkey kidney or other suitable
continuous cell cultures, such as baboon kidney, is
the preferred method where the lacilily is available.
Inoculated cell cultures an- incubated without scrum,
and in the presence o f trypan, which increases
sensitivity of isolation. Incubation at 33 'C in roller
drums is recommended. Virus growth can be
identified by hemadsorption with human O group,
fowl or guineapig erythrocytes. Rapid results can
be obtamed by demonstrating virus antigen in
infected cell cultures bv immunofluorescence.
3. Serology: Complement fixation and hem­
agglutination inhibition costs are employed for the
serological dingnosi- of influenza- It is essential to
examine paired sen in parallel, to demonstrate rise
in Liere of antibodies.
Complement (beat iontestswiththe R N P an1 L^en
of influenza virus types A, B and C arc very useful
as the antibodies are formed during infection only,
and not following immunisation with inactivated
vaccines. Complement fixation can also be done
using V antigens for the demonstration of strain-
specific antibodies. Because oJ its complexity, C F
tests are now used only tarelv.
Hcmagglutination inhibition is a convenient and
sensitive test for the serological diagnosis of
influenza. However, it has some disadvantages. As
the antihctnaglutinin antibodies arc su b ty p e-
specific, it is necessary' to use as antigen the strain
currently causing infection. The may ir drawback is
the frequent presence in the sera o f nonspecific
inhibitors of hemagglutination. The sera, suitably
treated for the removal of nonspecific inhibitors,
aie diluted serially in hemagglutiiur ion plates and
the influenza virus suspension containing 4 H A units
added to each cup. Fowl red cells are then added.
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separate. and also to keep pigs away from them.
The practice nt keeping several specie* n f birds
a Song w ith c h ic k e n ) in liv e tu rd m a rke rs is
potentially dangerous.
A unique feature ufinfluienza epidemiology ^a>
that once an antigenic valiant emerged, it displaced
completely the pre-existing strain. T hus when A l
( H lN l) strains arose in 19*16-47. they became the
o n ly viruses causing hum an disease, and the
previous AO f K flN I) strains disappeared completely.
The A l strains were displaced by Asian (112N2)
strains in 1957 and they; in rum, by the A2 Hong
Kcrng (H3IN2) strains in 1968. However, this rule
has not been observed in recent yeans. Even after
the irc'cmcigence and wide dissemination o f the
HIN1 strain in 1977, the A2 Hong Kong H3N2
strains continue to he prevalent.The rea-son for this
coexistence is roc known,
There is considerable evidence to suggest that
there occurs an o rd e rly recycling o f rhe viru s
subtypes at Least with regard to their hemagglutinin
(H ) antigen, be roepid cm to Logical (seroareheo-
logi-qal) studies indicate that the severe pandemic
of 1889 was caused by a virus with rhe antigenic
structure H2NE and that this was followed in 1900
by the subtype IL3N8 which led to a moderate
pandemic. Itl 1918 came [L]e most severe o f all
pandetpios, caused by the 'Swine type' H lN l
(formerly Hsw N l) virus. Mild epidemics occurred
around 1933 and 1946 associated with minor
variations in the H antigen (from Hsw to HO in
1933, H O lo H i in 1 9 4 6 ). The next severe
pandemic came in 1957 with the H 2N 2 (Asian)
suhtvpe. This was followed in 1968 hy the H 3N 2
(Hong Kong) virus leading to a moderate pandemic.
The year 1977 saw the reappearance of the H lN l
virus. Thus rhe sequence o f variation in the H
antigen has heen H 2—* H 3 -* H i —> H 2—> H 3 —►
H i from 1889 to the present time (Table 55.3),
FVtim 1977, both H3N2 and H lN l viruses have
been circulating together. Table 55-3 lists the
sequence of appearance o f these various subtypes.

1889-1900 H2 NS? Pandemic and epidemics

1900-1910 H3 N8 Extensive epidemics
1919-1933 Ht Nl (former Hm Nil 'Spanish flu'- The must severe pandemic
recorded; Heavy mortality.
1 9 3 3 -1 9 4 6 HI Nl (former H0N1) Discovery ofinHutniui virus
(VVS (train-1933);Epidemict o f strains
1946-1937 HI Nl Epidemics ot"LA l' strains
1957-1968 H2 N2 Extensive pandemics of Asian flu' formerly
calkd A2 (.Asian) strain, low mortality.
1968 H3 N2 Moderate pandemic ofHong Kong flu'
TOthe present time fnnneiLy called A2 rime (Hong Ktmg) strains,
very low mortality.
1977 HI Nl Re-emergence of former A l strains. First
cu the present time appeared in kussia and China ('Red flu );
Mild pandemic, very low mortality.

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 Paramyxoviruses
The family Pram yxoviriike contains important
p.Trh<vircns of infants .mil] children, responsible for
a major pant of acute respiratory infections
(respiratory syncytial vims and parainfluenza
viruses) and alwv fnr two of the most contagious
diseases o f childhood (measles and mumps).
Though much less common, infections may also
occur in adults.
Paramyxoviruses resemble orthomyxoviruses in
morphology but are huger and more pleamoiphiC.
They arc roughly spherical in shape and range in
size from 100 to 300 nmh sometimes with long
tilaments and giant forms of upto S(X) tun. The
helical nudeocapsid is much wider than in
orthomyxovi Rises, with a diameter of 18 nm (except
in fVieLrruunj-LTS where it is 13 Urn). The geiuitite is
a molecule of linear single stranded RNA. Unlike
the orthomyxoviruses, in which the segmented
nature o f the genom e facilitates genomic
rcassortrnents and antigenic variation so typical of
Influenza viruses, the paramyxoviruses with their
unsegmented genome do not undergo genetic
recomhmadoiLS or antigenic m iltf o a i Hence all
pantmyxovi ruses arc artigenic&dv stable.
The nudeocapsid is surrounded by a lipid
envelope which has rhe matrix (M) protein at its
baie and rwo types of trartsmembraiie glycoprotein
spikes at rhe surface. The longer spike is the
hem agglutinin (H ), which may also possess
neuraminidase (N) activity and is hence known as
H or HN protein. It is rcsptmsible for adsorption
of the virus to the host cell surface. The second
spike is the F (fusion) protein, responsible for fusion
of the viral envelope with the plasma membrane of
the host cells, which is the essential early step for
infection. It also brings about cell-to-cdl fusion,
causing large giant cells or syncytia, which are
characteristic of paramyxovirus infections. The F
protein abci mediates the hemolytic activity of
paramyxoviruses.
The faniiIy Paramjatoviridae is divided into lour
genet a— /fu 5uJ;e i i rus Fa rai n flu e n zu vj’ru s,
AJarfjJLVinjs and PVicLutMnms (Table 56,1),
Mumps is an acule infectious disease commonly
affect i ng children and ch aracterised by
nonsuppurative enlargement of the parotid glands.
As epidemic parotitis, it had been described by
Hippocrates in the fifth century BC. The viral
etiology of mumps was demonstrated by Johnson
H id Goodpasture ( l 9.14) by its experimental
transmission to monkeys-1 label in 1945 cultivated
the virus in embryonated eggs. In 1955, Hcnlc and
Deinhardt grew it in tissue culture.
T h e mumps virus is a tvpicil
paramyxovirus possessing hoth HN and V proteins.
It agglutinates the erythrocytes of fowl, guineapig,
humans and many other specioa. I lemagglutination
is followed by hentolysis and elution at 17 ' C. Tlie
virus can be grown in chick embryos - in the
amnionc cavity for primary isolation and the
allantoic cavity alter adaptation. Eggs are inoculated
at b-B days and incubated at 35 °C lor five days
before harvesting.
Cell cultures are better suited for isolation -
primary monkey kidney being the preferred cell.
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 Human viruses PuninfluMM 1-4 Mumps Meades Respiratory syncytial
virus
Diameter of nudeocapsidlnm} 19 IB IS 13
Fusii m (F) protein t + + +
Hcmofysin + + + -
] Icmagplminin/Hcmadsorption + + t -
Neuraminidase + + - -
InrraeclllLdar inclusions in C C N,C C
cytoplasm {C)/ nucleus (N)

The cytopathic effect is stow and consists o f F.pididymo-orchitis is a complication seen in

syncytium formation and the presence of acidophilic
cytoplasmic inclusions Growth is host identified
by hemadsorption.
The mumps virus is labile, being rapidly
inactivated at room temperature of by exposure to
tormaldehvde, ether or ultraviolet light. It car. be
preserved at —70 "C or by lyophilisation.
The mumps virus is antigcnically stable and only
one serotype exists. Two complement fixing antigens
can be recognised, is in influe naa viruses— the
soluble (S) antigen and [he Viral' (V ) antigen.
Infection is acquired by
inhalation, and probably also through the coniunctivu.
The vims replicates in the upper respiratory tract and
cervical lymph nodes and is Jissen iiuaied through
the bloodstream to various organs. The incubation
period is long, about 12—25 days,
Parotid swelling is usually the first sign of
illness though it may sometimes be preceded by
prodromal malaise. Parotid swelling is unilateral
to start with hut may become bilateral. It is
accompanied by fever, local pain and tenderness but
the skin over the gland is not warm or erytliemacous*
Parotitis is nonsuppurative and usually resolves
within a week. However, involvement of the
evtr.iparotid sites may be more serious. Such
involvement tn^y sometimes occur even in the
absence of parotitis.
about a thind of postpuherta! male patients. The
tesfis hecomes swollen and acutely painful, with
accompanying fever and chiUs, Orchitis is usually
unilateral but when it is bilateral and followed by
testicular atrophy, sterility or low sperm counts may
result.
The central nervous system is involved in about
60 per cent o f cases* as indicated by pleocytosis in
the C SFbut only about 10 percent show symptoms
of meningitis. Mump - has been reported to cause
about 10-1.5 percent of cases oTascprie meningitis'.
Mumps me itiiigi Lis and meningoencephalitis usually
resolve without sequelae but deafness may
som etim es result. M um ps m eningitis may
occasionally occur in the absence of parotitis, when
diagnosis rests solely on laboratory evidence, The
virus can be grown readily from tile C S F in the
early phase of meningitis.
Other less common complications arc arthritis,*
oophoritis, nephritis, pancreatitis, thyroiditis and
myocarditis.
Mumps is endemic worldwide
but has become less common in the advanced
nations due to immunisation. It often occurs as
epidemics In children 5 -1 5 years of age, and also
in young people living in groups such as in army
camps. Household spread is common. Humans are
the only natural hosts. The source of infection is a

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Parainfluenza type 1 Hemadsorption type 1(HA- 2)
Parainfluenza type 2 Croup iSSOCiaied (CA)
P*rain fluent* ty-pe 3 Hemadsorption type 1 (HA-1)
Parainfluenza rvpe 4 {4A 4li
hemagglutination inhibition,
Serological diagnosis is hampered by wide
antigenic cross reactions. Paired sera can be tested
by neutralisation* ELISA* HI or C F for rise in titre
of antibodies.
The Newcastle disease virus (avian paramyxovirus
type 1} is a natural pathogen of poultry in which it
causes explosive outbreak o f pncumoenccphalids
or ‘influenza with high mortality. In India it is
known as the Ranikhct vims. Control measures
consist o f vaccination* and slaughter of infected
birds.
Human infection with NDV is confined to a
self-limited conjunctivitis ill poultry workers and
others in contact with infected birds.
Other types o f avian paramyxoviruses cause
inapparent infection in many species of birds.
RSV was first isolated in 1956 from chimpanzees
with coryza and was called the ‘chimpanzee coryza
agent' (CCA ). A year later, the virus was ohtqincd
from children with lower respiratory'traer infection.
Because it caused cell fusion and the formation, of
foul tn undented syncytia in cell cultures, it was named
respiratory syncytial virus (R S V )r It is now
recognised as the most important cause o f lower
respiratory tract in tec nun in infants, particularly in
the first few months of life.
RSV is pleomorphic and ranges in size from
P--'h, W ~~b~\
Sendai (mouse)
Simian viruses 5, 41 (monkey)
Shipping fever (cattle)
1 5 0 ^ 3 0 0 n(ti. T h e viral envelope has two
glycoproteins— the C protein by which the htus
attaches to cell surfaces, arid the fusion (]■’} protein
which brings about fusion between viral and host
cell membranes. The F protein is also rcspoisiblc
lor c e ll- t o -cell fusion, which leads tc the
characteristic syncytial cytopathic changes in RSV
infection.
RSV differs from other paramyxoviruses .n not
posseting hemagglutinin activity It also decs out
have neuraminidase or hemolytic pro parties.
Anot her difference is that its nuclcocapsid diuneter
(13 nm) is less than that of other paramyxcriruscs
(13 nm).
RSV does not grow in eggs hut tan he
propagated on hetenoploid human cell culture, such
as H l'L . l and H ep -3. It is highly labile and is
inactivated rapidly at mom temperature. Itcan be
preserved by lyophilisation, It is antigenicalV stable
and only one antigenic type exists. However.studies
using monoclonal antibodies have identified two
subtypes called A and B.
M ost RSV infectitns are
symptomatic. The virus can hardly ever h found
in healthy persons.
Infection causes a broad range of respiratory
illnesses. Ill infants* the disease may begin a febrile
rhinorrhea, with cough and wheezing, progressing
in 2 3-40 per cent to lower respiratory involvement,
including tracheobronchitis, bronchi edit h and
pneumonia. In about one per cent* the iilics.s is
serious enough to requite hospitalisation. tSV is
considered to be responsible for about half ilu cases
of bronchiolitis, and a quarter nf all pneumonias
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occurring in the first few month? of life. Most
pat [cuts recover in 1 -2 weeks but those with
immunodeficiency ur cardiac defects mav have
protracted illness and high death rates.
RSV’ is ail important cause of otitis media in
young children. A relation between RSV and the
sudden death syndrome in infants has been
proposed bur not proven,
In adults RSV infection may present as a febrile
common cold. It can cause pneumonia in the elderly.
RSV is global in distribution.
It causes annual epidemics in the temperate regions
during winter and in the tropics during rainy
seasons. Outbreaks are common in childrens wands,
nurseries and day care centres. Infection \s most
common in children six weeks to six months of
agef with die peak at 2 -3 months. The newborns
are believed to be protected by high Levels o f
maternal antibody
The virus is transmitted by dose contact, and
through contaminated finger? and fomites. Coarse
droplets of respirator secretions discharged during
coughing and sneering are more efficient in
spreading the virus than fine aerosols- The
incubation period is 4 -6 days. Virus shedding may
persist for 1 -3 weeks, though children with
defective cell mediated immunity may continue to
sited the virus for months.
Reinfection with the virus is not uncommon but
the disease so produced is milder than in primary
infection. The role of the antibody in protection
against the infection is not clear- Secretory IgA is
considered more importune than circulating IgG
in protection. Ceil mediated immunity appears more
important than humoral antibodies in itonvery from
infection. RSV does not induce high levels of
interferon production.
RSV can he isolated
from nasopharyngeal swabs or nasal washings,
Samples should be inoculated in cell cultures
{H eLa or H E P -2 ) immediately after collection.
Freezing of clinical samples may destroy the virus.
In cultured cells, RSV causes characteristic giant
cell and syncytial formation but cytnpathic effects
take about 10 days to appear. Earlier detection of
viral growth in cells is possible bv immuno-
fiyorescence test?- Rapid diagnosis of RSV infection
can be made by tile immunofluorescence test Oft
smear* o f nasopharyngeal swahs.
Serological diagnosis is by demonstration of
rising antibody litres in paired serum samples by
ELISA, CFnneutralisation or immunofluorescence
tests.
N o effective vaccine in Available.
Attempts at immumsmion by fbrmaJinised vaccines
had to be given up as the vavcineeis developed inure
serious illness than the controls on subsequent
exposure to flic infection.
Management of RSV infection is
primarily by supportive Care. Administration of
rilrovirm by continuous aerosol has been found
beneficial In hospitalised patients, decreasing the
duration of illness and of virus shedding.
Measles is an ancient disease hut for a long time
no clear distinction was made between measles and
other exanthematous diseases, including smallpox.
It was only in ;162V that measles came to be
considered a separate entity. Thomas Sydenham in
1690 gave the first dear and accurate description
of measles in the English language.
In 1E146 an outbreak of measles occurred in the
remote Faroe Islands, affecting 75 per cent o f the
islanders. The classic study of this epidemic by Peter
Pamm i. a Danish medical student, laid the bash of
our scientific knowledge about measles.
The vital etiology of measles was established
bv G oldherger and A nderson in 1911 by
transmitting the disease to monkeys through the
inoculation of filtrates o f blood and nasopharyngeal
secretions from patients. The virus was isolated In
monkey and human kidney cells by Enders and
iVcblts in 1954.
T h e virus has the general
C o p y rig h te d m aterial
rttorphology at paramyxoviruses {Fug. .56.2) It is a
roughly spherics] but often pleomorphic particle*
1 2 0 -2 5 0 nm in diameter. The rightly coiled helical
nudeocapnid is surrounded hy the lipoprotein
envelope carrying on its surface hemagglutinin (I J}
spikes. The envelope also hast the F protein which
mediates cell fusion and hemolytic activities. The
measles vim* agglutinates monkey eiyihrocytes but
there is no elution as the virus does not possess
T te ijra m in id a H e activity.
The measles virus grows well on human or
monkey kidney and human amnion cultures which
are the preferred ceils for primary isolation. Isolates
can he adapted for growth on continuous tell lines
(H cLa, Vcro) and in the amninric sac of hen's eggs,
CyropathJe effects consist of muliinueleate syncytium
formation, with numerous acidophilic nuclear and
cytoplasmic inclusion*. Multinucleatr giant cells
(Warthin-Finkeldey cells) are also found in the
lymphoid tissues o f patients.
The virus is labile and readily inactivated by
heat, ultraviolet light* ether and formaldehyde. Jt
can be stabilised by molar M gSG+t. so that it resists
heating at 50 "‘C ior one hour.
The measles virus is anrigcnically uniform. It
shares antigens with the viruses of canine distemper
and bovine rinderpest.
It take* about 9-11 days
from the rime of exposure to infection fnr the first
signs of clinical disease to appear. These consist of
prodromal malaise, fever, conjunctival injection,
cough and nisjl discharge. After 23—4 days of
prodromal illness, the rash appears. A day or two
before the rash begin s, Koplik's spots develop on
the buccal mucosa and occasionally on the
conjunctiva and intestinal mucosa,The prodromal
illness subside* within a day or two o f the
appearance o f the rash. The m l maculnpapular ras-h.
of measles typically appears on the forehead first
and spreads downwards, to disappear in the same
sequence 3 -6 days later, leaving behind a brownish
discolouration arid fmety granular desquamation.
Most patients recover uneventfully but quite a
few develop complications which may be due to
the virus (croup, bronchitis) or to secondary
bacterial infection (pneumonia*otitiu media/. Karel1,-,
the virus may cause a fatal giant cell pneumonia,
particularly in children with immunodeficiencies
or severe malnutrition, Complications art more
common and serious in the developing countries.
The m oit serious com plication it
meningoencephalitis. M any survivors have
neurological sequelae. A rare kite Complication is
subacute sclerosing puicncephillds (SSPE).
P rotracted diarrhea is often seen as a
complication in children in the poor nations. The
virus may he recovered from the stnob of patients
with measles enteritis.
T h ere occurs a suppression o f delayed
hypersensitivity after mfades infection, which may
las! for weeks or a few months. Manloux and Other
allergic skin rests may be negative during this period.
Underlying tuberculosis may becom e worse
following an attack o f mcisler. Recovery from
measles may also he associated with an
im provem ent o f allergic eczem a or asthm a,
Hodgkin's disease or lipoid nephrosis.
Measles induces labour in some pregnant
wlat virus* 1.
gaptltf 1 - r-r ini
: : t ; 1 kiln.
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Hidden page
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 Arboviruses
A A m i ruR« Iarthr-npnd home v l t u r c s ) arc v i m s e ?
of vertebrates- biologitally transm itted by
hematnphagmuR insect vector?. 1 Hey muttiplv in
bloodsucking insects and are transmitted by bite to
vertebrate hosts. Insert viruses and viruses of
vertebrates th at are sometimes mechanically
transmitted bv injects do not come into this
category. Inclusion in this group is based on
ecological and epidemiological considerations and
Hence i t contains memben; that arc d i s s i m i l a r in
other pmpcrriL-s. W ith better understanding of the
physical and chemical properties of individual
virusesf they arc reassigned to more defined
t axo no ft 11 cal groups. T h ou gh taxonom ically
unacceptable, the name ‘arbovirus' ]S a useful
hiological concept.
A similar ecological group is that of the
rodentbome (robo) viruses which axe maintained
in nature by direct transmission between rodents,
and sometimes infecting other species, including
humane, by direct contact without the agency o f
arthropod vectors. Robovi ruses, Iike arboviruses,
belong to d tferent taxonomical families, some of
them in common with arboviruses.
Artovinjscs arc worldwide in distribution ban
arc far mote numerous in the tropical than ir. the
temperate zones. Over 500 vinues have been listed
in the International C itilo g u e o f A rboviruses
published lil 1965. M ost of them cause silent
infections in rodents and other wild mammals but
about 100 o f them can infect humans. In India,
over 40 arboviruses have been detected, o f which
more chan 10 are known to produce human disease.
Arboviruses have been named according no the
disease caused {Yellow fever), the place of initiation
of the virus (Kyasanur Forest Disease) or the local
name for the disease (Chikungunya), They arc
classified according to their physical and chemical
features- into taxonomic al families. Arboviruses have
been placed in Toga-, Flavi-, Bunya-, Reo- and
Rbabdovjrus families {Tabic 57-1). Within each
family, they ore classified into genera, and antigenic
group*, based cm serological relationships. Some
viruses arc ungrouped-
Arbnviruses have a very wide host range
including many jpccic? of animals and birds. The
ability to multiply in arthropods is their special
characters ic. The moil important arbovirus vectors
are mosquitoes, followed by ticks. PhelbotOWUS,
Cufjcoirfprand Cim/p’dac arc less common vectors-
In rhe laboratory, mice are commonly used for
growing arboviruses, intracerebral inoculation in
suckling mice being the most sensitive method for
their isolation. They can be grown in the yolk sac
or chorioallantdic membrane uf chick embryo, in
Tissue cultures of primary cells like chick embryo
fibroblasts or continuous cell lines like vero or
I IcLa, and In cultures of appropriate insect tissues.
Most ilbwuMws aggluiinate the red cells of
goose or day-old chicks. Hemagglutination is
influenced by pH and temperature, the optimal
requirem ents varying with different viruses.
Spontaneous elution does not occur.
Hemagglutination in inhibited specifically hy
antibody, and nonspecific ally hy lipoprotein
inhibitors in serum, brain and other tissues.
In general, arboviruses are labile, being readily
inactivated at room Temperature and hy bile salt?,
C o p y rig h te d m aterial
 Toguvir idae Alptiavirus Chikungunya, O'nyong-nyn-ng, Mayam, SemlLki Forest
Sindhis, Riws River, Eastern, Western and Venezuelan
equine encephalitis viruses

Flftviviridw Flavivinu Japanese encephalitis, Murray Valiev encephalitis, WesrNjle,

Uheus, Sr. Louis encephalitis. Yellow Fever, Dengue types 1,
2, 3. J, Russian Spring Summer encephalitis complex,
Louping ill, Fowassan, Kvasanur Forest Disease. Omsk
hemorrhagic fever

Bunyaviriidae Bunyivims California encephalitis, Oropouclie, Turlock

Phleboviras Saniltlv (ever vinisrs, Kitt valley fever virus-

Nairovirus Crimean Congo tiemorrhagie lever viruses* Nairobi sheep

disease vims. Canjam vims

Hantavirus Hantan, Seoul, PViumala, Prospect Hill, Sin Nnmhre viruses

RL’ - I > v i c L l L i L c: Orhiviru-i Colorado tick fever; African horse tidiness, Blue rongue
viruses

Rhabdoviridae Vesiculovirus- Vesicular stumatitis vims, Chandipura virus

ether und other lipid solvents. Infccrivity may be
retained at -TO or bv lyophilisation.
Three antigens; are
important in serological studies - hemagglutinins,
complement filing and neutralising antigens - all
integral parts of the vims par tide. Considerable
antigenic cross reactions occur among aibovimses.
The plaque reduction neutralisation test (PR N T)
shows the greatest specificity.
T h e virus enters the body
tiirough the idle o f the insect vector. A fter
multiplication in the reticuloendothelial system,
vircmiin o f varying duration ensues and, in some
cases, the virus is transported to the target organs,
such as rite central nervont system In
ennephalLtides, the liver in yellow fever and the
capillary endothelium in hemorrhagic fevers.
Arboviruses cause the following clinical
syndrom es: fever with or w ithout rush and
arthralgia; encephalitis; hemorrhagic fever; and the
characteristic systemic disease, yellow fever (Table
57~2 )■ All infections occurwith varying degrees of
severity, subclinical infections being very common.
Arboviruses also cause a number o f veterinary
diseases iuch as Eastern* Western and Venezuelan
equine encephalitis in horses in America, Rift
Valley lever in sheep and cattle in Africa, Blue
tongue in asses in India, Africa and America,
Ganj am d isease of sheep i n 1ndia and African horse
sickness in horses and mules in Africa and Asia.
D iagnosis may he
established hy virus isolation or scmlngy. As all
arbovirus infections arc viremic, blood collected
during the acute phase of the disease may yield rhe
virus. Isolation may also be made from the C S F in
some encephalitic cases but the best specimen for
virus isolation is the brain. Specimens are inoculated
intracerebrally mto suckhng mice. T he animals

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Hidden page
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 « Arboviruses »
teaser extent in complement fixation testa. The
neucrilisatii m test is more specific, Huey produce
epidemics of encephalic in America and dengue-
iike fever i.n the tm|[iics.
E n cep h alitis v iru ses: Three members o f this
groups Eastern, Western and Venezuelan equine
encephalitis viruses, cause encephalitis in horses
and humans. Eastern equine encephalitis (E E E )
OCCiirs along eastern C anada, U SA and the
Caribbean, causing sporadic cases and small
epidemics. Western equine encephalitis (W E E ) is
more widely distributed in America and causes luge
epidemics. Venezuelan equine encephalitis (V EE),
prevalent ilL Central and South America, usually
causes an influenza-like illness, with encephalitis
in a small proportion o f cases. Several species o f
Culcx and Anopheles mosquitoes are the vectors,
and wild birds the reservoirs. FormaliniEcd vaccines
have been developed for E E E and W E E and a
Live attenuated vaccine for V EE,
V iru s c * C a u s in g F e b r i l e I lln e s s
I. Chrfaingunya wrttfjThis virus was first isolated
from human patients and A ed es leg y p ii
mosquitoes from Tanzania in 1952- The name
'■chikungunya' is derived from the native word
for the disease in which the patient lien "doubled
up* due tD severe joint pains. Epidemics of
chjkungunya have occurred i l many African
countries. In 1 9 5 B, the virus caused a large
epidemic of hemorrhagic fever In Thailand. The
virus first appeared in India in 1963, when along
with dengue, il caused very extensive epidemics
in C alcu tta, M adras and other areas.
Chikungunya outbreak's occurred at irregular
intervals along the east coast of India and in
Maharashtra till 1973. Since then the virus has
been quiescent.
The disease presents as a sudden onset of fever,
crippling joint pains, lymphadenopathy and
conjunctivitis, A maculopapular rash is common
and some show hemorrhagic m&nifestaiions.
Hemorrhagic lesions were common in Calcutta
525
when the disease first appeared there in 1963
hut have been extremely rare afterwards. The
fever is typically biphasic with a period o f
remission after 1—6 days of fever. The vector is
Aedei- itegypli. Nt> animal reservoir has been
identified. Antibody to the virus has been
demonstrated in horses, cattle and other domestic
animals but its significance is not known. No
vaccine is available.
2. f) uyong-nyongYirui'- This virus was first isolated
in Uganda. This is confined to Africa, is closely
related to the ehikungunva virus anrigcnically
and causes a similar disease. This is transmitted
by the Anopheles species. The Mayaro virus
causes a similar disease in the Mfest Indies and
South America.
3. Semi.K: Forest vi;v£ This virus was. first isolated
in 1942 in Uganda from Aedes mosquitoes has
not been associated with clinical illness in
humans though neutralising antibodies to the
virus have been demonstrated in Africans, The
Sindh is virus, originally isolated from Culcx
mosquitoes in the Sindbis district of Egypt in
1952, has subsequently been recovered from
other puts of Africa, India, Philippines and
Australia. In Africa, it is known to be associated
with febrile illness in, human beings. In Indi^
antibodies have been detected in human sera
but no association has been established with
human disease. The closely related Ross River
virus has been associated with epidem ic
polyarthritis in Australia.
F U V W 1RUSES
The family Flaviviridae contains only one genus,
FfavivifuS. They are somewhat smaller than
alphaviruKs, being 40 nm in diameter. The name
Flaviviws refers to the type species, the Yellow fever
virus (ivJavLrs, L = yellow).
There are over 60 arthropod-borne flavivimaes.
Representative members a f this group are
distributed in all parts of the world* covering all
the zoogpogjaph ic regio ns. They may be considered
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Hidden page

In India, I.L p a n e s e
recognised in 1955 when the vims w u isolated
from mosquitoes of the Ciiibr raYh/iuj complex from
Vellore during an outbreak of encephalitis in Tamil
Nadu, The vims continued to be active in Tamil
Nadu and Andhra in subsequent years also, causing
illness mainly in children, indicating the endemic
nature of the virus. Most of the cases occurred
between October and Nawanbcn
J i pine sir CMcun hah Ln rem .Lined confined to the
southeastern parts of Indii.i till 1973, when it caused
a large outbreak of encephalitis in West Bengal,
The epidemic affected adults also, with mortality
rates approaching 50 per cent, suggesting that the
virus was freshly introduced into the area. Cases
occurred mainly between June and October. Prom
1976, there have been periodical outbreaks of the
disease in various parts o f India— Dibrugarh
(Assam) in the cast, Gorakhpur (Uttar Pradesh)
,h11 lLEl.uvai i .lm the north and G o, land Maharashtra
m the west. In the south, outbreaks have occurred
in Kolar in Karnataka, various areas in Andhra
Pradesh, I’irunelveli and South A rent in Tam il
Nadu, in Pondicherry and lately in Kerala. In
addition, sporadic cases have been reported from
different parts n; the country, excepting the
northwestern states. Japanese encephalitis has
become a major public health problem of national
importance in India (Fig. 57.1).
The natural cycle of the virus has been worked
out in derail in Japan. Herons act as reservoir hosts
and pigs as amplifier hosts. Human infer!iinn is a
tangential 'dead-end' event and occurs when the
i n f e c t e d n 1 1 :■ ■ ■ li ■.j 11 ■ -L" ■ ■ i l . ll j i
lYlIl1In India also may be similar. Natural infection
has been demonstrated in Andeid birds (herons and
egrets), as wdJ as bird-ft f-b iid transm'issi' )n thr*>ugh
C-’tiJcv mVaen/orhync/ius, Other binds such as ducks,
pigeons and -sparrows may also be involved.
Vertebrate hosts may Include cattle and buffaloes,
besides pigr. T h e m;\ii>r v ector C ulcx
tritpfniarhynchus has a predilection for cattle and
bites them in preference to humans or pigs, but as
encephalitis was first
t n j^ k J i i ji s i 1 y . " T T i t
« ArtHYlTlJ&BB I 527
cattle do not develop ri:cra u, they do not contribute
to spread of the virus. The high cattle- pig ratio in
India has been suggested as a factor limiting human
infection.
Preventive measures include mosquito control
and Ineat] iLg p iISeries away from human dwellings.
A formalin inactivatcd mouse brain vaccine using
the Nakayama strain has been employed successfully
for human immunisation in Japan and, in a small
scale, ] ii India also. Two doses at two weeks interval
followed by a booster 6 -1 2 months later constitute
a full course. Immunity produced by the vaccine la
shortlived. A live attenuated vaccine has been
developed m China from J E strain SA 14-14-2,
passed through weanling mice. The vaccine is
produced in primary' baby hamster kidney cells,
Administered in two doses, one year apart* the
vaccine has been reportedly effective in preventing
clinical disease.
Vaccination of pigs has been proposed in view
of their importance as amplifier hosts. During
major epidemics, slaughter o f pigs have been
employed as a measure of containment, A million
pigs were reportedly slaughtered in Malaysia in
1999 to stop an epidemic of encephalitis.
Yellow fes ll]t Yellow fever was recognised as a
clinical entity as early as in the seventeenth century
and was familiar to pirates as the 'Yellow Jack'. Jr
is a native of Africa and was transported thence
along the trade routes to Europe and America.The
most serious epidemics occurred in the Western
I lem [sphere- Centra I America and the Caribbean,
and even as far north as New York. Since the early
i i i l U l t .i I i w c n i i i - t l i . Lui - . i rv. t h e i L i l-l■ k . r ; K e e n l a r g e l y
confined io certain areas of Alriea and South and
Central America.
Carlos Finlay in Cuba in 1331 suggested that
yellow fever was spread by A cd es aegypti
mosquitoes. In 1900, the US Army Yellow Fever
ComnU'Kiopi, underw riter Reed, confirmed this
observation and dem onstrated th a t Aedes
mosquitoes were infected by feeding on human
pari ents during the early vi remi l: phase of the disease
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Hidden page
 * Arboviruses ►
,l: kI became infective after an extrinsic incubation
period of 12 days. This led to the prompt eradication
of the disease from Cuba and the Panama Canal
area by control] mg the Aedes aegypti mosquitoes.
There were even hopes o f ultimate total eradication
of the disease but these had to be abandoned when
in 1932 outbreaks of yellow fever occurred in Brazil
Ln areas devoid of Aedes aegypti, It was then
recognised that the virus survives in another cycle
- the forest or sylvatic cycle - involving forest
animals and mosquitoes.
The yellow fever virus was first isolated in 1927
by inoculating rhesus monkeys with the blood of
an African patient named Asibi. T he virus was
shown by TWiler {1930) to grow well following
intracerebral inoculation in mice. T he infected
mouse brain was used as a vaccine in former French
W est A lrici (Dakar vaccine) though thus was
cnccphalitogcitic. It was later replaced by a non-
ncurotropic (17D) vaccine
After an incubation period of 3 - 6 days, the
disease starts as a fever o f acute onset with chills,
headache, nausea and vomiting. The pulse is usually
slow despite a high tem perature. Jaundice,
albuminuria, und hemorrhagic manifestations
develop and the patient may die of hepatic nr renal
failure. Most cases are less severe, especially in the
endemic areas, and may present as undifferentiated
fever without |aundice.
His to logically, the liver shows- cloudy and fatty
degeneration and necrosis which is typically
mitizonah The necrosed cells coalesce and become
hyalinised leading to the formation of chaiacteris tic
eosinophilic masses known ns Councilman bodies.
Acidophilic intranuclear inclusion bodies (Tomes
bodies) may be seen in the infected liver cells in
the early stages. The histological picture o f the liver
is specific enough to be diagnostic, and this was
the basis of early surveys undertaken to detect ire u
o f yellow fever activity, A special instrument
(viseerotome) was employed for the collection of
liver tissue from fatal cases for histological
diagnosis.
529
The epidemiology of yellow fever was clarified
only after the recognition that the disease occurs
m two distinct patterns. In the urban cycle, humans
at both as the natural reservoir and as the definitive
case, the virus being transmitted by the domestic
Aedes zegjpti mosquito. In the forest or sylvatic
cycle, wild monkeys act as the reservoirs and forest
mosquitoes [Hacmagvguz spzgazzinii in South
America and Aoc/es ifriianus and A. yrrtJpsOJt; in
Africa) as the vectors. Human cases occur only
when humans trespass into the forest or when the
monkeys raid villages near the forest.
T h e control o f urban yellow fever can be
achieved by eradicating the vector mosquito, as was
shown in Cuba and Panama early thin century by
Gorgas but this is obviously impracticable with the
sylvatk disease. Two very effective vaccines have
been developed for human use. The French
ncurotrOpi c vacci rue (Dakar) pr<jduced ffortl infected.
mouse brain was thermostable and administered by
scarification and hence convenient for use under
tropical field conditions- However, the vaccine
carries a high risk of producing encephalitis in the
vaccinees, especially in children. A safe and equally
effective vaccineh the 17D vaccine was developed
by Theiler in 1937 hy passaging the Asibi Strain
serially in mouse embryo and whole chick embryo
tissues and then in chick embryo tissue from which
the central nervous tissue has been removed. The
17D vaccine is t he nliu labile1and is administered
by subcutaneous inoculation. Vaccination which is-
mandatory for travel to or from endemic areas is
valid for 10 years beginning 10 days after
vnecinafion, Tn India, the 17D vaccine ■*
manufactured at the Central Research Institute,
Kasauli.
Yellow fever is largely confined to Central and
South America and Africa, Yellow fever does not
exist in India and it is important to us for rhis.
paradoxical reason. India offers a reoptiivcarea with
a Luge population o f Aedes aegypti and nommmune
humans. Strict vigilance is enforced on vaccination
and quarantine for travel from endemic areas.Thin-,
C o p y rig h te d m aterial
530 4 Textbook o1 Microbiology ►
no doubt, has checked the entry of the vi™* Into
India through legjtirmtepasscngei^.Ir is likely that
stray virus introduced may have been kept out, due
to the prevalence in the local A cJ h aegypti of
] fengue virus, and o f antibodies to a wide range o f
arboviruses in the local population. Another reason
could have been that in Africa, yellow fever was
mainly in the west, and in Indii.L, Aeries mosquitoes
were along the east coast, so that even stray
importations of virus by sea may not have found
suitable vectors. Thfr in no longer valid as yellow
fever has in recent years have caused epidemics in
East Africa, and Aedes mosquitoes have spread all
along the went coast of India. If ever yellow fever
gets established in India* the consequences could
be catastrophic.
D en g u e: Dengue virus in widely distributed
throughout the tropics and subtropics. (The name
"dengue' derived from the Swahili Kj deilg* p£pQt
meaning a sudden seizure by a demon. The term
'break-bone fever’ was coined during the
JJhiladelphLa epidemic in 1790). Dengue fever is
clinically similar to the illness caused by
chikungunya and O'nyong-nyong viruses. Four
types of dengue virus exist: D EN 1 first isolated
from Hawai in 1944, D EN 2 from Nrw Guinea in
1944 and D EN 3 and 4 from the Philippines in
195A. Immunity Ls type specific so that it in possible
for a person to have lour separate episode^ of dengue
fever. Dengue has been increasing worldwide over
the last few decades and today ranks as the most
important wetorborne disease, with about 2.5
bi 11ion people in 200 countries at risk.
Dengue presents clinically after an incubation
period of 3 -1 4 Aiy., as lever o f sudden onset with
headache, retrobulbar pain, conjunctival injection,
p-nn in the back and limbs (break-bone fever),
lymphadenopathy and maculopapular rash. The
fever is typically biphasic (saddle.back) and lasts
for 5 -7 days. Dengue may also occur in more serious
forms, with hemorrhagic manifestations {dengue
Hemorrhagic fever) or with shock (dengue shock
syndrome]. These complications, first recognised
in Thailand, have since occurred, in many countrius
in Southeast Asia and the Western Pacific. They
are mote common in previously healthy children.
In the indigenous populations of endemic areas.
They may be a hypersens itwity or enchancement
response to sequential dengue virus injection in
persons sensitised by prior exposure to other
serotypes of the virus.
Dengue virus is transmitted from person to
person by Aedes a ^ p t i mosquitoes. The extrinsic
incubation period is £ -1 0 days. No vertebrate hosts
other than humans have been identified.
Dengue was initially confined to the east coast
of India and has Caused e pi.Jc rciits, some tirues along
with the chikungunya virus, as in 1963 when
extensive outbreaks affected Calcutta and Madras.
Subsequently it has spread westwards and in the
1990s Sunt and Delhi had major epidemics with
deaths due to D H F and DSS. All four types of
dengue virus are present in this country.
Occasionally, more than one type of the virus has
been isolated from the same patient.
Demonstration of circulating lgM antibody
provides early diagnosis, as it appears within two
to five days o f the onset o f illness and persists for
one to three months. IgM E L IS A test offers
reliable diagnosis. A scrip imnnunochromaiographic
test for IgM is avaitable for rapid diagnosis.
Control o f dengue is limited to vector control
as no vaccine is currently available.
T ic ib o r n b G ro up
These v'irases produce two clinical syndromes,
encephalitis and hemorrhagic fevers.
T i o k b o m t en cep h alitis v iru ses: A number
nf viruses belonging to the Rusiiui Spring Summer
Encephalitis (RSSE) complex cause encephalitis
along a wide area ot the northern lindm.iiH from
Scotland to Siberia. The names given to the disease
vary from one area to another depending on the
variations in the prominent clinical features. Thus,
in Scotland, it is called 'louping ill' as the disease
occurs primarily in sheep in which it c b u e c & a
C o p y rig h te d m aterial
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This, family containing over 300 species in the
largest group of arboviruses. The virus is about 100
rtm in diameter and has a complex structure, with
a triple segmented genome of single stranded RNA.
Most bunya viruses are mosquirobomc. Some ire
transmitted by sandflies (for example, Phlebntomus
fever) or ticks (Crimean Congi t hemorrhagic fever).
Some are established pathogens, causing natural
diseases, and even epidermcs and epizootics, while
many hive been isolated only from insect vectors
and have not been associated with any tauman or
animal disease. BunyaiuruscH ate ho named from
the type species Bunyamwera virus isolated from
mosquitoes in Uganda in 1946.
The family Bunyaviridae contains four genera
of medical importance— Bunya virus, Phlebovjirus,
Nairovirus and Hantavirus. A number of viruses
are yet ungrouped.
Tile genus contains over
150 species, o f which only a few cause human
in fection s. The clinical disease caused is
encephalitis, aseptic meningitis and fever. The
California encephalitis group o f viruses are endemic
in the USA. Large epidemics of fever with aseptic
meningitis have been caused by Qropouchc virus
(member of theSimbu group} in Brazil-The midge
C ulicoidtt /^araenf/a is the major vector for the
Oropouche virus.
The major members o f
this genus are the sandfly fever and Rift Valley fever
viruses.
FAidtoiumus fever or sandfly fever, also known
as Pappaiuci fever and three-d.av fever, is a self-
limitcd, nonfatal fever transmitted by the bite of
the sandfly PA/eicromus papufusn. It occurs along
ih t M editerranean C oast and C entral Asia,
attending as far east as Pakistan and North Went
India. Cases have also been reported from South
and Central America. Twenty antigenic types o f the
virus cjcj h t , o f which only five cause human
disease —'Naples, Sicilian, Punts Toro, Chagres,
Candiru-Tbc virus has been isolated from sandflies
and patients in India. No t c deb rate host other tlian
humans has been identified. Theft is evidence for
vertical transmission of the vims in sandflies.
Rf/r Valley fev er in a mosqiutoborne virus
causing enzootic hepatitis in sheep and other
domestic animats in Africa. It is named after Kitt
Val1eyr, Kenya, where it was first recognised. Human
infection causes a disease resembling influenza. In
1977-EQ , R ift Valley fever caused extensive
epidemics, with many deaths in Egypt and an
outbreak nl hemorrhagic fever with many deaths
in 1997-98 in Kenya. In 2000, it spread outside
Africa for the first riuie, causing e |ndem ics in Ycmen
and Saudi Arabia with hundreds of devths-
The genus is named after
C o p y rig h te d m aterial
 h Arboviruses *
rhc type species Nairobi Sheep Disease Virus,
Memhen; of the Crimean Congo hemorrhagic
group are the major human pathogens in this genus.
Fhe- Crimean hemurrhagii fever virus, hir-' isolated
.11 Crimea in 1945, was subsequently found to be
idertLicit with the Congu fever virus isolated in 1956
in Congo {Zaire), hence the name Crimean Congo
Hemorrhagic Fever (C C H D + T h e disease is
endemic in E i i t c m Europe, Central Asia and many
parts of Afuca. Cattle, sheep, goats and other
domesticated animals act as natural reservoirs. It is
transmitted by Hyalomma ticks, During the acute
phase of the disease, the blood of the patients is
h ■i^KI) in feet ious and direct Tranwn issit in may occur
through contact. A related virus, Hazara, has been
isolated in Pakistan. It is also widespread in Iran,
Iraq and the UAE- Antihtsdie* to the CCH F group
af viruses have been detected in human and animal
sera from India.
Nairobi sheep disease is an acute, hemorrhagic
gasiroenreritif caused by a Niorwirus in sheep and
gnats in East Africa. It is transmitted hv
Rliipicqib.ilu- ticks. The virus produces a mild
febrile illness in shepherds tending infected flocks.
The Ganjam virus, isolated from ticks collected
from sheep and goats in Orissa, India, is closely
related to rhc Nairobi sheep disease virus. The
Ganjam virus has also been isolated from human
sources. Accidental infediun in laboratory workers
has caused mild fehrile illness.
(iiniijh Hantavirm : This virus causes
hemorrhagic fever ^ich renal syndrome (HFRS),
also known as endciric or epidemic
nephrosoncphrlris, \1 atich ur ia n epidemic
hemorrhagic fever, nephrnpathia epidemic a.
rodenf-home nephropathy and other names. This
condirlon first attracted attention in the earlV 1950a
when i large number of US soldiers serving in
Korea got the infection but it has been prevalent in
Scandinavia, R ubsLi and China Mr centuries.
The disease occurs in two formH— the milder
epidemic nephritis (EN) common in Scandinavia
and the more serious epidemic hemorrhagic fever
533
(E H F ) In the far east, The eiini cal picture resembles
typhoid, leptospirosis and scrub typhus.
The genus contains at leant :our sp ecies—
Hantaan vims caus-ing the severe HFft$ in the l- .Lr
East, North Asia and Russia, Scotif virus causing a
m ilder type o f d incase aod probably present
worldwide, Puuniala virus responsible for
ncphnopatKia epidemics in Northern and Eastern
Europe, and Prospect Hill virus isolated from voles
in the USA, which has nut been associated with
human illness.
Hantavirus species are natural pathogens of
rodents— field mice {Apodeitim agntr/us) being the
major host for Hantaan, rats f/fcftus rantis and ti.
norvie^jcui) for Seoul, and voles for PuumaJa and
Rmspect Hill viruses. Vircmia is present in infected
rodents and the virus is shed in, urine, feces and
saliva in high ritrcs. Transmission from rodent to
rodent and rodent to humans is primarily
respiratory, by inhalation of the virus contained in
dried excreta. Domestic rats appear to he the source
of infection in urban cases HFRS-Though there
are reports of the role of mites in the transmission
of the infection, this is not confirmed. In the
absence of proved arthropod transmission, HFRS
should be cnnsiilered a robovinis and not strictly
an arbovirus infection.
Demonstration of IgM antibody by ELISA or
of rising litres of immune adherence
hem agglutinating antibodies- in laired -icra are used
for laboratory' diagnosis.
A new syndrome, the HuntHriruS pulmonary
syndrome was identified in south western USA in
1993. After a prodrome of fever, malaise, myalgia
andgastytiinfestinal Ryi npft 11 ns, last ingfor days,
patients develop pulmonary involvement with carle
radiological picture of pulmonary edema, but few
physical findings. In severe cases, tachypnea,
tachycardia, hypotension and hypoxia lead to death.
The disease is caused by a newly idenrifled
hantavirus, the Sin Afombre {meaning nameless''
virus, which is associ.itrd with the deer mouse and
other rodents of the sigmodonviue subfamily. No
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 h R tobdovkuses
TTtfUt cutout:"] III rabies virus cm grow in several
pTimaiy and continuous cell cultures such as chick
embryo fibroblast, porcine or hamster kidney bur
cytopafhi.- effects are not apparent and the yield of
vi.us is low. The fixed virus strains adapted For
growth in human diploid cell, chick embryo and
veto cell cultures are used for the production of
vaccines.
RABIES
Rahir' - has been rreognised from wry ant ient :i mes
as a disease transmitted to humans and animals by
the bite of'mad dogs'. The name 'rabies’ comes
from the I .it in word nabnJus, meaning mad, derived
tom the SiJi'-krU root rafe/ris, fur feemy. Reference
to rabies occurs in the Mesopotamian law? of
Fshnunna (£ rirca 220G BC), The disease was
traditionally associated with the appearance of the
Dog Star Sirius in the 4dog d.ivs af summer when
dogs were considered to be prone to spells of
'madness*. The disease in human beings is called,
hydrophobia because the patient exhibits fear of
water, being incapable of drinking though subject
to intolerable thirst. Rabies in animals is not called
hydrophobia, because they do not have this peculiar
feature.
The causative agent of rabies had, for centuries,
been associated with the saliva of rabid dogs but i.t
was only in 1304 that Zinks adduced proof by
transmitting the disease to normal dogs by the
inoculation of saliva from rabid dogs. In 1321,
Magendic and Brcschet infected dogs with saliva
from a human patient, proving the identity of the
agent causing human and animal rabies. To a series
o f studies dating from 1KE1, Pasteur established
that the rabies virus was present in the brain of
infected animals. By serial intracerebral passage in
rabbits, he obtained the fixed virus and demons traced
that dogs could be rendered immune by a series of
injections of fixed virus of graded infectiivity. This
vaccine was prepared by drying for various periods
pieces of spinal cord from rabbits infected with the
fixed virus. In July 1995, Joseph Mrifter, a nine-
t 537
^car-i>Ld boy+severely bitten by a rabid dog and in
grave risk of developing tabirs, was given a course
of 13 inoculations of the infected cord vaccine by
Pasteur. "Ilie boy survived. This dramatic event was
a milestone in the development of medicine.
PuthojJjcncsis: Human infection is usually
caused by the bite of rabid dogs or other animals.
The virus present in the saliva of the animal is
deposited in the wound. If untreated, about half of
such cases may develop rabies. Rarely, infection can
also occur following non-bite exposures such as
licks or aerosols or transplantation of cornea or
other virus infected tissues. Humans appear to
possess a high degree of natural resistance to rabies.
The extent of inapparrnl or abortive Infection with
rabics virus in humans is not known but the finding,
in a survey, of rabies antibodies in six per cent of
veterinarians without any history of antirabic
vaccination suggests that it does occur.
The virus appears to multiply in the muscles,
connective tissue or nerves at the site of deposition
for 48-72 hours. It penetrates the nerve endings
and travels in the axoplasm towards the spinal cord
and brain. The movement of the virtue m the aborts
is paHi-ivc, at a speed of about 3 mm per hour. The
infectii>n spreads centripetally from the axon to the
neuronal bodice, and progressively up the spinal
COld tli rough the ^napses of the neurons. The vi Cut
ascends rapidly to the brain where it multiplies and
spreads centrifugally along the nerve trunks to
various parrs of the body including the salivary
glands. It multiplies in the salivary glands and is
shed in the saliva. The presence of the virus in the
saliva and the Irritability and aggression brought
on bv the encephalitis ensure foe transmission and
survival of the virus in nature. The virus ultimately
reaches virtually every tissue in foe body, though
the centrifogal dissemination may be interrupted
at any stage by death. The virus la almost invariably
present i n the cornea and the feci;J skin of patients
because of their proximity to the brain. This
provides a method for the antemortem diagnosis
id human rabies. The virus may also be shed in
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538 i Tyxiboe* of M icr LJSio ogy
m ilk and urine. Viren in is not clinically significant
though if has been demonstrated under ocperiiriental
conditions.
In humans che incubation period is :j -ll:l11v from
1-.1 months, though it may be as short as 7 days or
as long as three years. The incubation period is
usually short in persons bitten on the face or he.id.
and long in those bitten on the legs. This may be
related to the distance the vims has to travel to
reach the bruin. The incubation perW is generally
shorter in children than in adults.
The course of the disease in humans can be
classified into four stages— prodrome, acute
enccphald ic phase, coma and death. The omet is
marked by prodromal symptoms such as fever,
headache, malaise., fatigue and anorexi.L. An early
symptom is often a neuricic type of pain or
paresthesia and fascieularicm at the site of virus
entry. Apprehension, anxiety, agitation, irritability,
nervousness, insomnia or depression characterise
the prodromal phase, which usually lasts 2-4 days.
E x ce ssiv e libido , pri .Lpi sm and sp u n tan eo us
ejaculation may occur rarely
The acute neurological phase usually begins
with hyperactivity, which is characteristically
intermittent, with bouts uf bizarre behaviour,
agitation or seizures appearing between apparently
normal periods. Such hyperactivity may be
spontaneous or pier ipirated hy external sti muli.The
pathognomonic feature is difficulty in dririkiug,
together with intense thirst. Patients may be able
to swallow dry solids but not liquids. Attempts to
drink bring on such painful spasms of the pharynx
and larynx producing choking o r gagging that
patients develop a dread of even the sight or sound
of water (hydrophobia), Generalised convulsions
follow. Death usually occurs within 1-6 days due
to respiratory arrest during convulsions.
Some patients progress to paralysis. In rare cases,
hyperactivity may nor be prominent and paralytic
features dominate from the beginning. Such
paralyse disease i-, more common in Latin America
and Trinidad after exposure to vampire bat rabies,
*
and in those who have received postexposure
vaccination. Patients who survive the stage of acute
neurological involvement lapse into coma, which
may last for hours or days. Death is due to
respiratory arrest or other complications.
Some persons exposed to real of imaginary risk
of rabies develop a psychological disorder which
has been called tyssaphohia or hydrophohlophobia.
Patients present with anxiety, irritability and
exaggerated hydrophobia, They arc afebri Ic.
Sedation and reassurance are generally all that are
called ft>r.
In dogs, the incubation period is usually 3-6
weeks but it may range from 10 days to a year. The
initial signs are an alert, troubled air and a change
m disposition with restlessness, snapping at
imaginary objects, licking or gnawing at the siie of
the bite. After 2-3 days of this prodromal stage,
the disease develops into e idler the furious or dumb
type of rabies. In filrioua rabies, which Is much
more common, the dog runs amok, biting without
provocation and indisc rLiinnately. The lower uw
dmgps and saliva drools from the mouth. Paralysis,
convulsions and death follow. The second type,
dumb iahics, is the paralytic form In which the
animal lies huddled, unable to feed. The dog may
not nice but attempts to feed it are dangerous. The
dumb form 1e as infectious as the furious type. About
60 per cent of rabid dogs shed the virus in saliva.
Rabid dogs usually die in 3-5 days.
There have been reports of persons developing
rabies after being bitten by apparently healthy dogs.
However, in countries like India where stray dogs
arc eo common, it in not always c.lkvto exclude the
possibility of such patients having been bitten earlier
by other animals. The presence and prevalence of
rabies vims carrier state in dogs hat been a matter
of controversy and concern. If ar all, this is a very
rare event. The possibility of carrier dogs has not
so far altered the recommendations fbr postotposwc
treatment.
Rabies in cats is similar to canine rabies, hchnc
rabies is an important source of human infection m
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 n RhnUdoyiruFieF; ►
I.Ltnir.Llijji-l ill India manufacture BPLvaccuie.
3 . Zntimt bi.nii vjiccjr)es. The cncephalitogenlc
factor in brain tissue Ika basic protein associated
with myc'-in, It h scanty or absent in the
nonmyelinated neural tissue of newborn animals.
So vaccmes were developed using infant mouse,
rat or rabbit brain. Occasional cases of
neurological reactions have occulted following
infant brain vaccines also- Infant brain vaccine
is impractical in India due to the very large
quantities required.
Neural vaccines are unsatisfactory for many
reasons. "I’hcy arc poor immunogens as they contain
mostly nuclcocapsid antigen, with only small
quantifies- of glycoprrU^ii: t which is the sole
protective antigen- They may contain infectious
agents which may not he inactivated during vaccine
preparation and storage. They are encephalitogenic.
Neural vaccines have been abandoned in the
developed countries. The oitlv reason for their
continued production and use in a few developing
countries is that they are cheap.
N oN -N EutiA L V a c c i n e s
1 £ & race-lies: (a) Duck egg racvnie prepared
from a fined vims adapted for growth in duck
eggs and ireacti vated wi th beta prupiolaetone was
used, but wps discontinued because ul its poor
immunogenic ify, A purified, more potent duck
egg vaccine was developed, but was supplanted
by tissue culture vaccines which became
available then, (b) Live attenuated chiick embryo
vaccines: two types o f vaccines were developed
with the Flury strain-rhe Low Egg Passage
(L E P ) vaccine at 40-50 egg passage level for
immunisation oi dogp and the High Egg Passage
{H EP) vaccine at ISO passage level for cattle
and cats, these are nor in use now.
2- T j UIK! cu/nifl VMC.'MS: The first cell culture
vaccine was the human diploid cell (H D C )
vaccine developed by Koprowsky, Wiktor and
Plotkm. It is a purified and concentrated
preparation of fixed rab'i^ virus {Pitman-Moore
S41
strain) grown on human diploid cells (W I dfl
or MKL 5) and inactivated with beta
propiolactonc or tri-n-butyl phosphate. It is
highly antigenic and free front serious side
effects. Tts only disadvantage is its high cost.
Other equally effective and more economical
vaccines have been developed. Ib csc include:
prim in' cell culture vaccines grown on chick
embryo, hamster kidney and dog kidney cedis and
COrJr.'Jiunus cell culture vaccines grown on Veilu
cell line derived from the kidneys of vervet
monkey or African green monkey (Ccrcopit/icai.s
aerfn'npr).
In India the following cell culture vaccines are
available: Human diploid cell {H D C } vaccine
Purified1chick embryo cell (P C E C ) vaccine and
Purified verv cell fFVCJ vaccine. All three of
them arc equally safe and effective.
3, Submit raceme. The glycoprotein subunit on
the virus surface, which is the protective anugcn,
has been cloned and recombinant vaccines
produced. They arc still in the experimental
stage.
V accin a tio n S c h e d u i es
Antirabic vaccine should be administered when a
person has been bitten, scratched or licked by an
animal which is rabid or cannot be apprehended.
When the biting animal can be observed, it should
not he destrtjyed but should be kept for ten days.
The observation period often days is recommended
because the vims may be present in tllC saliva 3-4
days before onset of symptoms and the animal
usually dies within 5-b davs- of developing the
disease. If the animal remains healthy after this
period* there is no risk of rabies and vaccination, if
already started, tnay be discontinued-Thin, of course,
does nor take into account the rare possibility of
the carrier state in dogs.
In cases where the vaccine is started with the
hiring .mimil kept under observation, an alternat ive
recommendation is to stop treatment after five days.
The animal is observed for a further five days*
C o p y rig h te d m aterial
*4 2 i Tejiiboe-; nf Micro oio'ogy *
vaccine being starred again if the animal becomes
ill a r dies during the period.
N t LIT ill VACdilLtK The dosage of the vaccine
depends on the degree of risk to which the patient
has been exposed. Accordingly, pat ients are classified
as follows:
Chttt I: Patents in whom the risk is estimated to
be slight. These include:
a. Licks, incluiling direct contact with saliva on
definitely remembered fresh cuts or abrasions
on all parts of the body except the head, face,
neck or fingers.
b. Licks on intact mucous membrane or
conjunct! va.
c. Hites or scratches which have rai-sed the epidermis
but have not drawn blood, on all parts of the body
except the head, face, neck or fingers.
d. Consumption of unboiled milk or handling raw
flesh o f rabid animals.
Class II: Persons who are estimated to be at
moderate risk. These include:
a. Licks on definitely remembered fresh cuts or
abrasions on the fingers-,
b. All bites or scratches on the fingers which are
not lacerated, not more than half a centimetre
long and have not penetrated the true shin.
c. Hites or scratches on all parts of the body except
the head, face, neck or fingers which have drawn
blood but excluding bites which have five teeth
marks or more, or in which extensive laceration
has occurred.
Class III: Persons in wham the risk is estimated
to be great. These include:
a. Licks on definitely remembered fresh cuts or
abrasions on head, face or neck.
b. All hues or scratches on the head, face ur neck.
c. All bites or scratches on the fingers which are
lacerated, more than half centimetre, long ur
hive penetrated the true skin.
d. All bites penetrating the true skin and drawing
blood, when there are five teeth marks or more.
C, All hites on any part of the body causing extensive
laceration.
f. All jackal and wolf bi-es.
g. Any Class 11 patient who has not received
treatment within 14 days of exposure.
The rra>mmcndcd schedule of vaccination frw
the different classes is as follows:
Semple vaccine P P L vaccine
Class I 2 ml x 7 days 2 ml * 7 day
Class I I 5 ml x 14 days ml 10
3 i lI . i v :-;
Class III 10 ml x 14 days 5 ml x 10 days
The above schedule for the BPL vaccine is
recommended by the Pasteur Institute, Coonoor.
The Central Research Institute, Kasauli,
recommends a slightly different dosage for its
vaccine, fThe manufacturer;; r.'isnucflonsshould be
followed in every rasel
The immunity following vaccination with
neural vaccines is expected to last far six m onths
only and any exposure later should receive fresh
treatment. The vaccine i$ administered
subcutaneously on the anterior abdominal wall.
Anti tali iC Vaccine may cause certain adverse
reactions. These range from minor local reactions
to serious neuroparalytic complications. The latter
may he of the neuritic type, the dorcilumbar type,
the Landry type of ascending paralysis, or
encephalomyelitis. The etiology of neurological
complication is believed to be immune response to
the injected brain : issue resulting in organ specific
immunological damage .lh in experimental ullcrgic
encephalomyelitis. These complications usually
occur within 1 -4 weeks of commencement of
vaccination, The incidence of complications varies
with different vaccines, one in 2000 to one in 12,000.
When such complications are noticed during the
course ol vatvination, further vaccination should
be withheld and the p&ricnt slatted on
corticosteroids. If further vaccination is considered
imperative, non-neural vaccme should be used.
Severe exertion and the use of alcohol during
vaccination have been said to increase the risk of
neurological reactions.
Coll cu ltu re vocables: All three cell culture
vaccines avuJable in India (HDCi PC E C. and PVC)
C o p y rig h te d m aterial
have the same dosage schedule, which i* the same
for both adults and children.
Pre-exposure prophylaxis requires Three doses
of iltt vaccine injected on day 0, 7, 21 or 0, 28 and
56. A booster dose is recommended after One year
and then one every five years,
Fostexposure prophylaxis requires five or six
doses, on days 0, 3, 7, 14, 30 and optionally 90.
This course is expected to give protection for at
least live years-, during which itcriod any further
exposure may need only one or two fimnsier doses
(on days 0, 3) depending on the degree of risk.
After five years, it is advisable tin give a full five
injection course if exposed to infection.
The vaccine is to be given INI or SC in the
deltoid region, OF in children on rbe anterolateral
as]>cet nf the thigh. Gluteal injections arc to be
avoided as they are found to be less immunogenic.
It has been shown that a dose of 0.1 ml
administered intradermaily is as effective as a 0 .3 -
1.0 ml dose SC or LM and that immunisation may
thus he made more earn imural. However, this is
not recommended as a routine practice, as
iniradermal injection is technically difficult, and it
will be ineffective if this dose is given
subcutaneously by mistake.
Anlirabic serum is
manufactured by hyper immunisation of horses.
Crude equine antirabies serum is not to be used as
it is liable to induce anaphylactic reactions. Purified
equine rabies immune globulin (LRlG ) is much
safer, though not completely free from risk. Human
rabies immune globulin (IJRJG) is tret from the
danger of sensitisation but should be ensured free
from HIV and hepatitis viruses. HRIG is much
costlier than £R1G,
Passive immunisation is an important adjunct
to vaccination and should be invariably employed
whenever the exposure is considered of high risk.
The recommended dose of H RIG is 20 lUAg body
weight, half the volume infiltrated Jt the site of
the wound and the other half injected in the gluteal
region. Passive immunisation should be given before
or simultaneously with the first injection of" the
vaccine, but not after it. In person? receiving the
serum and vaccine, a booster dose of cell culture
vaccine on day 90 may be given.
Recommendations tor postexpoRure prophylaxis,
as endorsed by the W H O in 1988, are shown in
*Vkc£ioe failures' (persons developing rabies
even after a full course of immunisation) arc not
mi common with neural vaccines, while they are
i ;py righted m ateria
 ] Touching or feeding of .inidmk.
licks on intact skin
II Nibbling nf uncovered skin;
minor scratches or abrasions wit hour
hleeiling; Licks Oft broken akin
in Trans dermal b it« or scratches -
contamination of mucous membrane
wi th saliva
extremely rare when immediate Wal treatment ban
been followed by rabies immunoglobuliiii and a full
course of a cell culture vaccine. In view of the safety
of the cell culture vaccine, ]C would be advisable to
recommend the vaccine even when there is the
slightest risk of exposure to rabies.
An t irabies intnuirtisaLioiL
in animals is to be done as pre-exposure prophylaxis.
Postcxposure treatment is nut generally of much
use, Neural vaccines are not satisfactory as rhey are
not adequately imrnimogeok, need multiple doses
and have to he repeated every six months.
Concentrated cell culture vaccines containing
inactivated virus are now available, which give
good protection after a single IM injection.
Injections are given at \2 weeks of age and repeated
at 1-3! year intervals. Rabies vaccines mavbe given
sepsmitclvor as combined vaccine tor immunisation
against other common veterinary infections also.
Until recently, rabies was considered
to bo invariably lata! and iro serious attempt ar
treatment was made, apart from sedation. It has
now been demonstrated that Complete recovery can
occur from established rabies, with intensive
supportive care and management of complications.
No specific antirabies agent is available.
Human rabies is a dead end.
Direct person-to-person transmission of rabies has
None {if history is reliable)
Start vaccine. May be discontinued if
animal ia well after 10 days
Rahies immune globulin vaccine to he
started. May be discontinued if animal
is well after 10 days
not been recorded, though the virus is present in
the saliva of patients. Therefore, there is no danger
in examining or nursing hydrophobia patients
provided suitable precautions are taken. An unusual
mode of trantimlsskin of rabies hai occurred in some
recipients of corneal grafts. The donors had died
of unsuspected rabies mid the infect ion was
transmitted through the cornea.
Rabies virus is present in terrestrial animals in
all parts of the world except Australasia and
Antartka, and some islands like Britain. Two
epidemiological types of rabies exist, - Urban,
transmitted hv domestic animals like dogs and cats;
and sytvatic, involving animals in the wild, such as
jackals, wolves, foxes, mongooses, skunks and ban.
Most ciLses- of human rabies follow Jog bites but in
endemic areas almost any animal can transmit
rabies. In Indk, antirubic treatment in to he
considered following fhc bite of any animal except
rats. Where urban ■, r ji-mestic rabies has been
controlled, as in the USA, the majority of infections
are due to bites bv wild auirnals.
The primary source of ilie rabies virus in nature
seems to he in the mustel'ds and vtverrids, rhe
ermine in the northern coni (emus forests, the skunk,
mink and weasel in North America, the mottled
]hJ c cat in the USSR, the civet and pole ciT in
Africa and the mongoose in Asia. Rabies vims has
C o p y rig h te d m aterial
been isolated repeatedly from the brain jind salivary
glands of apparently healthy wild rodents. The virus
survives in thin reservoir population by achieving
a Stare of latency with occasional activation such
that only a snail proportion of the in will be
shedding the virus at any one time. From the
reservoir species, wild vectora such as frraea, wolves
and jackals acquire the infection, and occasionally
upiZoOlies occur in these species. Carnivorous
animals may acquire the infection by eating carcasses
containing the virus. From these species the disease
spreads to dogs and other domestic uni main.
A smouldering epizootic of rabies in. the red
fax in Europe had spread westwards steadily from
Poland to France during the last few decades.
Vaccine hit its (chicken head o r or her meat
containing live attenuated rabies virus) have been
used to immunise the red fox in an attempt to check,
the epizootic in the forests of Eurnpe.
Another natural cycle of rabies concerns hats.
A fatal paralytic disea.se of cattle and humans was
noticed in Central and South America and the West
Indies early in the twentieth Century. This was
identified as rabies only years Eater. The disease wat
shown to be transmitted by vampire bats that sweep
down on their prey at night. Vampire hat rahies
had talten a heavy toll of cattle. Vampire bats may
1 RahieS Warm blooded animals
2 Lagos bat/ Natal bat Bat/cat
3 MokoEa Sh rew/TAt/ciog/human
4 Duvenhage Humart/bii
5 European bat lyssavirus: Bat/humm
Type I
6 European bat lysEaviruti Bat/human
Type II
7 Australian bat lyss-aviras: Bat/human
shed the rabies virus as sympfomlcss carriers over
a period of several months.
Rabies also occurs in insectivorous and
frugivorouN bat Infection in insectivorous hats is
symptomatic, while frugivorous bats become
asymptomatic carriers-, While in canines rabies is
neurotropic, in bats the virus is primarily adapted
to the respiratory tract. Humans maybe Infected
by aerosols if they enter caves where infected hats
colonise. Pneumotropic rabies virus strains have
been oh rained from bats. Bat rahies is largely
confined to fhe Americas. A few strains of the rabies
virus have been isolated from bars in Europe but
their epidemiological significance is not known,
RahieS is endemic in India. It has been estimated
that more than 30,000 people die of rabies in India
every year and more than 700,000 receive ami rabies
vaccine. Human rabies can be checked hv control
of Tobies in domestic animals, by registration,
licensing and vaccination of pets and destruction of
stray animals. With the dog population in India
estimated as over 16 million, the problem is
immense. I lowever, rabies can be eliminated only
if the wild vectors such an jackals and foxes, and
the reservoir mustelidH and vivertids are controlled.
Rabies has been eliminated from islands like Britain
and Japan by rigid quarantine. Australia which has
Worldwide with few exceptions
Nigeria/Central and Knuih Africa.
Nigeria/other African countries
South Africa
Europe
Europe
Australia
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546 i TtH)e|booK Ol M.::mbiotofly ►

no native mustehd or bvcrrid population h;l> no
rabies. Eradication of rabies from countries Like
! ndi;t wi rh abundant wildlife may not be practicable.
RABIES RELATED VIRUSES
The genus Lyssavintsi consists ufthr values virus
and other serologically related viruses. Lyssaviruses
have been classified into seven serotypes.
1. Rabies virus is classified as Lyssanms serotype I
2. The Lagos bat virus, classified as LyssaviruL
serotype 2, was isolated in 1956 from the pooled
brains of frugivmous bats from Lagos Island,
Nigeria, It causes a rabies-like illness following
intracerebral inoculation. Negri holies are found
in infected monkey brain but not in mice or dogs.
3-. The MokuLa vinis, first isolated in 1968 from
shrews captured near Ibadan, Nigeria, has later
been found in many wild and domestic animals
in Africa, It was also recovered from two
children with central nervous system disease,
one of whom died. A case of laboratory infect ion
with the virus occurred in a person possessing
high titles of antibody to the rabies virus. It is
classified as Lyssavlms serotype 1
A. The Duvenhage virus was reported in J97l from
the brain of a man who died in South Africa of
clinical rabies after being bitten by a bar. It is
classified as Lyssavirus serotype 4.
5. 6. Rahies-Like viruses isolated from European
bats have been classified into two groups:
European hat lyssavirus types 1 and 2, They can
infect humans, as was found in the UK in 2002,
when a wildlife worker fell ill with 'rabies' and
died. This was the first "rabies1 death in the UK
in a century.
7. Australia was considered free of rabies and
related viruses till 1996, when a lyssatirus was
isolated from a frugivorous hat. Since then a
[lumber of similar isolates halve been obtained
from jLitfcrent types of bats in Australia. Fatal
infections have occurred in persons having
contact with bats. The virus antibody is widely
prevalent among Australian bats which appear
to be carriers. The virus, named Austrialrafl bat
fy'ssavifus is closely related to, but distinct from
the rabies viius. Antirabic vaccine and scrum
appear to protect against experimental infection.
The relevance of rabi c&related viruses in human
disease is not dear, though some of them have
caused illness and death in humans. They are
considered to represent a biological bridge between
the rakes virus and othcT rhabdoviruscs.

Further Reading
h u r GM. 1991. The Njiu fxl I h.-.icn o f Huhw-^, tio'h.n: CRC Press.
FiEhbc:ii BD ind LE RnbLnann 199.1 Halim, ,Vrw F-nglJ Med 329:1632.
Hupprrcht CE er si. 1994. Ly?±i viruses. Curwrlf Tiipw? in Microbiol Immunol 1B7.
Smith JS ct il. 1990. Unexplained rabies in three irtimlgiunTt in the United States. New EngJ Med 324:1990.
Smith J5. 1996. New aspects o f rabies, d in Mi&nbivt Rev 9:166.
WHO. 1992. Expen Cammirree art Rtfhfrs. Srb Report- Technical Series No. 924, Geneva.

Copyrighted material
 Hepatitis Viruses
The Derm 'viol heparin-s" refers to a primary Infection
of the liver by any one of a heterogeneous group of
'hepatitis viruses', which currently consists of types
A, B, C, D, E and G. (The designation "type F had
lieen proposed for j>putative virus helieveJ to cause
transfusion-associated hepatitis, distinct from type
A to E. But it proved to he a mutant {HRs) of type
R virus and not a separate entity Type F was
therefore deleted from the list of hepatitis viruses.}
Hepatitis viruses me taxonomicaJy unrelated.
Except for ty p e R w h ic h is a I > \ A v iru s a ll th e
others ait RMA viruses. The features common to
them me their hepatotropism and ability to ca.use a
s im ila r ictcxk illn e s s , r a n g in g in severity fr o m th e
Tinappaitnt to the fulminant fatal forms.
As alt types of hepatitis viruses cause a clinically
indistinguishable acute illness, their differentiation
is based on their serological and molecular markers.
Elepariiis may occur incidentally during many other
vital infections, such as with yellow lever, Lassa
fever, Marburg, EBhcytomegalo, herpes simplex,
varicella zoster, measles, rubella or coxsackie
viruses. These arc not included in the category of
viral hepatitis.
By epidemiological jind clinical criteria, iwo
types of viral hepatitis had been recognised for long.
One type occurred sporadically or as epidemics,
affecting mainly children and young adults, and
transmitted by the fecal-oral mute. This was called
j'n&cfhr or infectious hepadm, later termed type
A hepatitis. A second type of viral hepatitis,
transmitted mainly by inoculation was originally
observed in persons receiving strum inoculation
or blood transfusion. This had been given various
names such as tujmoitJguLU, smarm jaundice, serum
hepatitis (because of its association with human or
homologous antisera so commonly used for
prophylaxis or therapy early in the twentieth
centum1) and OS/Wfosfon hepstitj.t It was laler called
type fJ hepatitis.
For a rime it was believed that ah viral hepatitis
was caused by cither of the two hepatitis viruses,
type A accounting for all infectious hepatitis and
Type B for all |)oSt-transfusion or serum hepatitis.
However, with the development of techniques for
Identifying type A and type B viruses, if became
apparent that in many cases of infectious and post-
tTransfusion Hepatitis no evidence could he found of
infection with either type A or B viruses. It therefore
became evident that the clinical syndrome [if type
A or LS hepatitis could also be caused by one or
more other uncharaetcrised viruses. The term non-
A non-I? hepatitis was applied to this group. Soon
a type C virus was identified as causing many
transfusion-associated hepatitis cases. A defective
virus which depends on the helper functions r>f type
B vims was called delta or type D hepatitis viruses.
Yet another type of hepatitis transmicredby the focal-
O n il f m l l c , p r e v a l e n t i i l G s t l y in i h c d e v e l o p i n g
nations was found to be caused byrhepatitis E virus,
The sixth member of the group, hepatitis G virus
can also cause hepatitis, hut its role has not yet
been adequately understood.
Type A hepatitis (infectious hepatitis} is a subacute
disease of global distribution, affecting mainly
children and young adults.
C o p y rig h te d m aterial
Hidden page
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third of the; world's population is estimated to be
have been infected by hepatitis R virus (HRV).
About a quarter of them hecome HRV Harriers. A
quarter of these develop serious liver disease,
including chronic hepatitis, cirrhosis and primary
hepatic career. As there is in effective- vaccine
against HBV, hepatocellular carcinoma becomes
the only human cancer which is vaccine-
prcvencablc. The W H O estimates that HBV
infection cnusci more than a million deaths a year
worldwide.
The incubation period is
long, about 1-6 months, The clinical picture of
hepatitis R is similar Co that of type A, but it tends
to be more severe and protracted. The onset is
insidious and fever is not prominent. Extrahepatie
complications like arthralgia, urticaria and rarely
polyarteritis or glomerulonephritis may occur.
These are ascribed to circulating immune complexes
containing the viral surface antigen.
About 9 0 -9 5 per cent of adults with acute
hepatitis R infection recover within 1-2 months of
onset and eliminate the virus from the body within
about six months, remaining immune thereafter.
Mortality Is about 0.5 to 2 per cent, hut may he
more in post-transfusion canes. About \ percent of
patients, particularly those having simultaneous
della virus infection develop fatal Ruminant
hepatitis.
A proportion of cases Cl—10 per cent) remain
chronically infected- I'hey may be asymptomatic
carriers or may progress to recurrent or chronic
liver disease or cirrhosis. A few of them may develop
hepatocellular carcinoma after many decades.
The pathogenesis of hepatitis appears to be
immune mediated. Hepatocytes carry viral antigens
and are subject to antibody-dependent NK. cell and
cytotoxic T cell attack, In [he absence of adequate
immune response, HBV infection may not cause
hepatitis, but may lead to carrier start- Therefore
infant? and immurodetlcicnr persons are more
likely to become uytnptotuttk carriers following
infection.
HRV is a 42 ntn
BN A virus with an outer envelope and an inner
core, 27 r.:n in diameter, enclosing the viral genome
and a I)NA poEynnerue (Mg 59.2), Because of its
unique features. HRV is assigned to a separate
family /ejMdnavindae{hepatotn(ipic: 1>NA viruses),
which consists of two genera, <Jrthoheptdnnvt/irs
containing HRV as welt as the woodchuck and
ground squirrel hepatitis viruses, and
AhllejAildjiaiamS containing the Ptkjii duck and
grey heron hepatitis viruses. HRV' is fiepdlnarimj
type 1.
The discovety of HBV was serendipitous. In
1965* Rlumberg, Studying human serum Lipoprotein
allotypes, observed in the serum of an Australian
aborigine, a new antigen which gave a dearly
defined line oi precipitation with sera from two
hemophiliacs who had received multifile blood
transfusions. J his was named the Australia jr?£igci\.
By 1969 the 'Australia antigen' was found to be
associated with serum hepatitis. I lwas suWLyjeutly
shown to be the surface component of HBV.
Therefore the name Australia antigen was changed
to hepatitis B nrface antigen [HBsAg .
Ew#tae» (HU=*g)
NbCtoOGOpS'dCf.ru
IMBcAgl
QUAPiC*y«i«r*s*
Plus Strand " j DNA
Blrardja^K)nj#
 Under [he electron T[]it-n *erx from type
13 hepatitis patients show three h^ies of pirtides
(tigs, 59,3, 59.4), The most abundant form is a
spherical particle, 22 nm in diameter. The siCCOnd
type of particle is filamentous or tubular with a
diameter of 22 nm and tff varying length, These
two particles arc antigemcedly identical and are
surface components of HBV {liRsAg) which are
produced in great excess. The third type of particle,
tar fewer in number, is a double walled spherical
structure, 42 nm in diameter. This particle is the
Complete hepatitis B virus. It was first described hv
Dane in 1970 and so is known as the Dune particle.
The envelope proteins expressed on the surface
of the virion and the surplus 22 nm diameter
spherical ami filamentous particles constitute the
hepatitis R surface antigen. HRsAg consists of two
major polypeptides, ore of which is glycosylated,
ElHsAg exhibits antigenic diversity. It contains
two different antigenic components - the common
group reactive antigen ,j , and two pairs of tvpc
specific antigens J-yand itt, only one member of
each pair being present at a time, E-IBsAg a n thus
be divided into four major antigenic subtvpes: acta,
adr, Jyw and jjt . The subtypes do not seem to be
important in immunity because of the dominant
antigen a shared by all The subtype* breed true.
And the index case and contact* in an outbreak* , '■ i
have the same suhfypc. They show a distinct ■T.:ri
geographical distribution, bubtvpv ayvriscommon 1 IBcAg, though inimujiologically distinct, are coded
from West Asia through the Middle East, to il>r by the same gene.
Western and Northern India; adw is common in The nuclcocapsid encloses the viral genome
Europe, Australia and the Americas; adr :* consisting of two linear strands of DMA held in a
V ir v .iIc n L :u S-mLlh j i i lI J-..!-■ r I t l d u i m d th e f . i r I ’ .mf; Con
a r e o l a r (’ lion. One
g U f j [fir lp I (tine
s t r a n d s i p l u s

jot is very rare. A number of other surface antigenic
reactivities (ar x, K trj r n, g) have been reported, but
rot adequately studied.
Mild detergent treatment disrupts the viral
envelope and expose* the Cure ur nucleoc-apsid. The
intigen expressed on the core is called the /irptitis
II l-ojt juttgtt) (HRcAg)- A third antigen called
the hepatitis W c antigen (IllieAg) ia a soluble
noopaniculale nuclcocapsid protein. HBcAg and
Ktrand) is incomplete, so th.ir the DNA appears
partially double stranded and partially single
stranded. Associated with the plus strand is a viral
Ds\'A polymentse, which hits both DNA-dcpendeot
DNA polymerase and RNA-dependent reverse
transcriptase function*. This polymerase can repair
the gip in the plus strand and render the genome
hilly double stranded (Fig, 59.(5),
’["he genome has a compact structure with four

i
overlapping genes. ThtLS gene codes for the surface
antigen. It consists of the S region arid two Pre-S
regions, l hre-S2 and Pnc-Sl. The protein coded for
by the S region is called the S or major protein.
When translation begins from the Prc-$2 region,
the M or middle protein is formed. When the entire
gene from Pre-Si is translated, [he L or large
protein results- The L protein is present only in
the virion, while the M and S proteins arc found
in the circulating HBsAg particles also.
The C gene has two regions, C and Prc-C,
When the C region alone is translated, [he cote
antigen [HBcAg) is formed. HBcAg is assembled
as the nudeocapsid core particles. It is not secreted
and does not circulate in hlond, Hut can be
dem onstrated in hepatocytcs by immuno­
fluorescence, Antibodies to HRc, both IgM ami
TgG appear in blood. IgG antibody to HBcAg
persists in hlood tong after all other serological
markers have disappeared and so provides a useful
marker of prior infection with HBV. If translation
begins from the Prc-C region, the resulting protein
is HFteAg, a nun particulate soluble antigen
possessing a signal protein which enables ]L to be
secreted. Ir is therefore present in circulation. The
presence of HBcAg in blood provides a convenient
and readily detectable marker of HRV replication
and high inactivity,
The P gene is the largest and Codes for [he DNA
polymerase enzyme.The X gene codes for a small
rtcmp articulate pro rein (H B xA g), which has
tran sac rivaling effects or both viral and some
cellular genes. This leads to enhanced replication
of HBV, a- well as of some other viruses, such as
the human immunodeficiency virus. HRxAg and
its antibody are present in patients with severe
chronic hepatitis and hepatocellular carcinoma-
A few cases of infection by mutant viruses have
been identified. Two types of mutations have been
studied. One type, initially identified in
Mediterranean countries, presents as severe chronic
hepatitis, caused by pre-core mutants unable to
synthesise HBeAg. Those infected with precore
fltnea Region! Gene products
s S Major protein {5)
S+Pne-Si2 MidtHeproteinfM)! HBsAg
S-dVe-SlfltSl Large protein (L) J
c t: HhcAg
C+Pre-C HbeAg
p DNA polymerase
X h b *a k
mutants may he positive for anti-HBe and antl-
HBc. The second group of so-called 'escape
mutants' have been seen in some infants bom to
HBe-At; positive mothers, and in liver Transplant
recipients who had received combined
immunisation with anti-J IBV immunoglobulin and
vaccine. They show mutation in the common a
determinant of HBsAg, preventing them from
being neutralised by anri-HEiAg antibody. If such
mutants become more common, they may pose
problems in hepatitis B prophylaxis.
HBV replicates within hepanicytCs. Vital DNA
exists in the hepameyte nucleus in the free
ertraebromosonud state or integrated with the cell
chromosome- Replication resembles that seen in
retroviruses, in that DNA is synthesised from an
RNA template by reverse transcription.
HBV DNA and protein have also been identified
in extrahepatic sites such as boot marrow, spleen,
lymph nodes and circulating lymphocytes, but
apparently no damage is produced in these locations.
The significance of this cxtrahcparic presence is
not understood.
C o p y rig h te d m aterial
Hidden page
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of the high cusl of imported vaccine. Now Thai the
vaccine is manufactured in India, and is available
at lower cost, it should be possible to include this
in the national immunisation scheduler
Specific diagnosis of
hepatitis t! rests on the serological den lo iis Eration
of the viral markers. If ls therefore necessary to
undetstand the sequence of their appearance in
blood (Fig- 5^.6).
H R sA g ls the first marker to appear in blood,
after inhsiHon, being delectable ever before elevation
of transaminases and onset of clinical illness, It
remains in circulation throughout the icteric or
symptomatic course of the disease. In the typical
case, it disappears within about 2 months of the
start of clinical disease, but may sometimes last for
6 months and even beyond. When it is no longer
detectable, its antibody, anti-HBs appears and
remains for very long periods. Anti-HBs is the
protective antibody;
HRcAg is not demonstrable in circulation
hecauRe it is enclosed within the HRsAg coat, but
its antibody, jnti-HBc appears in serum a week Or
two after the appearance ufHBsAg. It is therefore
the earliest antibody marker to be seen in blood,
long before anti-1 [Be or anti-HBs. As anti-HBc
remains lifelong, it serves as a useful indicator of
prior infection with HBV, even after all the other
viral markers heenme undetectable. Initially, antu
HBc is predominantly IgM, but after about h
months, it is mainly IgG, Selective tests for lgM
or IgG anti-FI Rc therefore enable distinction
between recent or remote infection respectively.
HBeAg appears in blood concurrently with
HRsAg, or soon afterwards. Circulating FIBeAg
is an indicator of active intrahepatlc viral replication,
and the presence in blood of DNA polymerase,
HBV DNA and virions, reflecting high infectivity.
The disappearance of HBeAg coincides with the
fall of transminase levels in blood. It is followed by
the appearance of anti-HBe.
For the diagnosis of EFBV infection, detection
of H RsAg in blood is all that ordinarily necessary.
T h e simultaneous presence o f JgM a n t i - H B c
C o p y rig h te d m aterial
 556 * Texiboc* of Miemtooiogy ►
indicates recent infection and the presence of IgG
anti-HBc remote infection. Occasionally, when the
level Lht H BsAg lh t(K>law to be detectsble, diagnosis
has to be made by testing for IgM anti-HBc.
HBeAg provides information about relative
infer rivify. Its pretence denotes high mfectivi.lv and
its absence, along with the presence of anti-HBe,
indicates low inftcrivity, As it ns. invariably present
during acute hepatitis, its testing is indicated only
in chronic infection and carrier*.
The presence t>f anti'HBs without any other
serological virus marker indicates immunity
following vaccination. Table 59.1 shows the
interpretation of various serological patterns Ln
hepatitis B.
Like HEeAg, HBV DMA is also an indicator
of vital replication and inactivity. Molecular
methods such as DNAiDNA hybridisation and
PCR* it present used for HBV DNA testing ate
highly sensitive and quantitative. HBV DNA level
in scrum reflects the degree ofhral replication in
the liver and so helps to assess the progress of
patients with chronic hepatitis under antiviral
chemotherapy.
P rop h ylaxis! General prophylaxis consists in
avoiding risky practices like promiscuous sex.
injectable drug abuse and direct or indirect contact
with blood, semen or other body fluids of patients
and carriers. Health education, use of the disposable
syringes and needles, screening of blood, semen and
organ donors, haw all helped to an extent, but these
alone cannot eliminate the risk altogether,
particularly in the developing countries. The only
certain method appears to be universal
immunisation.
Both passive and active methods of
immunisation are available. Hyperimmune hepatitis
B immune globulin (H BlG) prepared from human
volunteers with high title anti-HBs, administered
IM in a dose o f300-500 i.u. soon after exposure to
infection constitutes passive immunisation. It may
not prevent infection, but protects against illness
and the carrier state.
Active immunisation h more effective. The first
vaccine introduced in 1982, was prepared from
pooled plasma o f healthy hum an carrierE with high
level antigcncmia. The 22 nm HBsAg particles
separated by ultracentrifugation were treated with
proteinase, urea and formaldehyde and used as the
vaccine. This was immunogenic, but became
unacceptable because its source was human
plasma, limned in availability and not totally free
from possible risk of unknown pathogens. It
continues to be used in some countries because it is
cheaper.
The currently preferred vaccine is genetically
engineered by cloning the S gene ofH BV in baker's
yeast. It consists of nongtyccuylated HBsAg particles
alone. It is given with alum adjuvant, IM into the
deltoid or, in infants into the anterolateral aspect of
the thigh. Gluteal injection is not recommended as
it mav result in poor immune response. Three dosci
given at 0, 1 and 6 months constitute the full course.
Saoconvertion occurs in about 90 pel cent of the
vaccine es, A special vaccine containing all antigenic
components of HBsAg (Pre-Si, Pre-52 and S) has
been developed, which gives greater gerocon version.
Serconvertiun can be cheeked by testing for anti-
HRs which is usually detectable for about 5 years.
Clinical protection is believed to last much longer
Booster doses are needed only for those at high
risk.
For nonimmune persons exposed to HBV,
combined immunisation is recommended. For
babies bom to carrier mothers, a single injection of
0.5 ml of HBlG given IM immediately after birth,
infollowed by the full course ofvactinc at a different
anatomical site, the first dose being given within
12 hours of birth. When HBlG is not available,
the vaccine given alone has been reported to provide
protection.
T reatm en t: No specific antiviral treatment is
available for acute HBV infection. Interferon alpha,
alone Or in combination with other antiviral agents
such as lamivudine and fameye lovir, has been
beneficial in some cases of chronic hepatitis. There
L5 no effective treatment for the carrier Stare, though
spontaneous re-solution takes place in some of
them*
Attempts to identify the group of'non-A. nan­
s'viruses hy experimental infection in chimpanzees
led to the discovery of hepatitis C vims (HCV). It
is now the commonest cause of past-transRision
hepatitis in the developed countries.
The incubation period is
long* 15—160 days, with a mean of 50 days. The
acute illness is usually mild or anicteric. Overt
jaundice is seen in about 5 per cent of patients only*
The important part in type C hepatitis is the chronic
Illness. About 50 to SO per cent of patients progress
to chronic hepatitis, with some developing cirrhosis
and hepatocellular carcinoma,
HCV infection is seen only in
humans. The source of infection is the large number
of carriers, estimated to be about 200 million
worldwide- In general the epidemiology resembles
chat of hepatitis 13.
Infection i$ mainly by blood transfusion and
other modes of contact with infected blood or blood
products. Injectable drug abusers, transplant
recipients and immunocompromised persons are at
■•.../S* '•i*p
+ IgM - -
t i* g — Lact/chrunk I EBY infection or carrier state*
* ■B lgG
■ IgM ~ +/-
" ■ IgG 4-/- */-
- ” “ + “ Immunity following HBV vaccine
high risk, Sexual transmission is probably less
important* Vertical transmission from, mother to
baby may take plsus.
Thc infection occurs throughout the world, with
carrier rates varying from 1™20 per cent. HCV
mfecdon is prevalent in India too, with an estimated
12.5 minion cases. A quarter of all chronic hepatitis
cases in India is believed to be due to HCV
infection*
The virus has
not been grown in culture, bur has been cloned in
Escherichia, eoli. H C V is a 50—6 0 ran virus with a
linear single stranded RNA genome, enclosed
within a core and surrounded by an envelope,
carrying glycoprotein spikes. H CV resembles
flavi viruses in structure and organisation, and has
been classified as a new genus Hcjwdvims in the
family Flaviviridac-
The virus shows considerable genetic and
antigenic diversity At least six different genotypes
and many subtypes have been identified, indicating
high m utability Some genotypes are seen
worldwide, while others are localised. Because of
this diversity there is little heterologous or even
homologous postinfectiem immunity in hepatitis C.
The standard method
of diagnosis is antibody detection by ELISA, The
Acute HBV infection, highly infeenuajs
highly infectious
Lete/chronic HBV infection Ot carrier State;
low infectivity
Seen wely in early acute 1 1BV infection;
infectious
Remote HBV infection^ infectivity nil m
very kw
C o p y rig h te d m aterial
558
antigens used art various structural and non^
structural proteins cloned in E, c'oil There have
been three successive generation? of such antigens,
intruduced Do improve sensitivity and specificity of
serological di.Lgnosi*. Even the third generation
ELISA currently in use, employing N S-5 region
protein and synthetic peptides becomes positive only
months after the infection and shows nonspecific
reactions. Confirmaliijn by immupoblot assay is
therefore recommended. In H CV infection
antibodies appear irregularly and latrhlimiting their
diagnostic utility.
Identification of HCV RNA in blood provides
more sen* irive and spec itic results wi tHin a few days
of exposure to HCV. Molecular methods like PCR
and branched DNA assay arc employed for the
purpose,
E’ rop h ylaxis: Only general prophylaxis, such
as blood screening, is possible. No specific active
or passive immunising agent Is available.
Treatm ent; Prolonged treatment wirh interferon
alpha, either alone or in combination with antiviral
agents like ribavirin has been reported to be useful
in some cases.
H P E D (DELTA) HEPATITIS
In 1977, RijMtto and colleagues in Italy identified
a new vital antigen in the liver cell nuclei of patients
infected with hepatitis Q virus. This lias been shown
to be due ro the hepirotropic virus Delis or
Hepatitis D Virus (H DV). Delta is a defective
RNA virus dependent on the helper function of
HBV for its replication and expression. Therefore,
iI h a s no ] ilJepenifei i
t L H t e n l_ ll and can survive and
replicate only as long as HBV infection persists in
the host,
HDV i<- a spherical, 36 nm particle with an
outer coat composed of the heparin? B surface
antigen surrounding the circular single stranded
RNA genome. Though it resembles some plant
viruses, such as viroids or satellite viruses, it has
been proposed to be classified in a new genus
J^fcffainrug, because of its special features.
^ Textbook of Microbiology t
Its mode of transmission is the same as for HBV.
Two types of infection arc recognised, coin/ccdon
and it^perirtfeciJort. In coinfection, delta and HBV
are Transmitted together at the same time. In
superitifcction, delta infection occurs in a person
already harbouring HBV, Coinfection clinically
presents as acute hepatitis IS. ranging from mild to
fulminant disease. Super infect ion usually leads to
more serious and chronic illness, with deterioration
of the underlying HBV infection. No association
has been noted between HDV and hepatocellular
carcinoma.
Delta antigen is primarily expressed in Ever cell
nuclei, where it can be demonstrated by
immunofluorescence. It is only occasionally present
in scrum. Anri-delta antibodies appear in serum
and can be idem ified by ELJ SA. The IgM antibody
appears 2 - 3 weeks after infection and is soon
replaced by the IgG antibody in acute delta infection.
However, in chronic infection, the IgM antibody
persists for years. Delta RNA sequences have been
cloned and DNA probes have been developed for
the jap id idem ificatuin of delta particles in
circulation. The woodchuck has been found to be
i suitable experimental model for the study of HDV
infection.
HDV is distributed worldwide but is more
common in certain endemic areas. In the
Mediterranean countries, where it is endemic,
infection u spread commonly by nonpcirutancous
mutes, cspccialy by close personal contact. In the
nonendemfo areas, such as Northern Europe and
North America, infection in more often through
blood and blood products and is commonly seen in
drug addicts and hemophiliacs. Introduction of
HDV into nonendemic area? where HBV infection
is common may lead to outbreaks of severe hepatitis
with high mortality.
No specific prophylaxis exists, bur immunisation
with the HBV vaccine is effective as HDV cannot
infect persons immune to HBV. Screening of blood
donors for HBsAg automatically limits blood borne
HDV infection.
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 z
TaWe 39,2 Viral hipaiilis ly p n : comparative features

A B C D E
Virus HAV, 27nm RNA, HBV, 47nm DNA HCV, 30 6Qnm HDV, 3£-37nm HEV,32-34nm RNA
Picom avirus (Hepadnavirus) RNA, FlavivirtiB Defective RNA Herpesvirus
{HcpUnvinu) (hepativima) Deltavirus

Mode? of infection Fccil-oul Percutaneous Pcrcutuiccius Percutaneous Fecal-Oral

Vertical, Sexual

Age Affected Children Any age Adull s Any age Young adults

4 Textbook of Microbiology *
Incubation 15-45 3CMS0 15-160 3 0 -ISO IS-60
Pei iod( days)

Onset Acute InsuluHii Intidiout Insidious Acute

Ilinn b Mild Occasionally seven Moderate Occasionally severe Mildt except in preg
nsnty

Carrier state Nil Common Present NiHonljr with HBV) Nil

Oncogenicity Nil Present specially after Present Nil Nil

nniunl infection

Prevalence Worldwide Worldwide Ptobably worldwide Endemic areas Only developing

Copyrighted materi

(Mediterranean,N.Europe, countries(India, Aej£

Central and N. America) Africa, Central,
America)

Specific prophylaxis lg bid Vaccine Ig bid vaccine Nit HBV vaccine Nil

ID
 e HepglHis Viruses ► 561

R ending
Alter Hf. 1997, Acute non A-E heparin* in the USA and the role erfhepatitis G virus infection, New Fud/Med 336:7'll,
British Medical Buljetin. I99lb Hepatitis 2:46
Brawn J L 1995. Hepatitis C. / In&ct 30:95.
Cnilier L (cd), lWfi. Hepatitis A and E TbpfcvAt H^soni \iiaobioiogy tnd A&rabraJ Infectu ms 9* edn., LondoniAmold,
Dienstag JL and KJ luefbacher 1999. In Acute Vital Hepatitis. Himson'is Principles o f Internal Medicine 14* edn. New
fade Me-Graw Hill
HolLnper FB and 17 Liiry2D01 HeptitK B vinia. In Fiddi Virology Ifol 2.4* ed. Philadelphia: Williams and Wilkirw,
Hoofna^JH. 19B?LType D {Delta) Hepatitis, J A nxr Med Assoc 2frl:132L
Krawczyniki K. 1993. Hepatitis E. H cp ato ^ l7:931
Lcrmr $M and DL Thomas 1597. Vaccines to prevent viral hepuitii. NewEngtJ AJeJ 336:196.
Maifleid ME end JR GcUsn 2QQ3. SuppnutHig Hepaticii B, N t^EngJ Med 348; 848.
Sarin SK anti AK Singal ted*). 1W6- Hepariris fl in India. New Delhi: CBS Polishers.
Van Damme Pet aL 1997. Integration of hepatitis B vuonarioo Lora national immtWrtatiofl pmgramnvci- HAIJ11-1:1033-

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 Miscellaneous Viruses
PAPQVAVIRU5E5
The term lFapova a sigEa indicating the names
□f viruses, included iit lIlil group! plpflloms Vitus
of human beings and rabbits, polyvma vims of mi;c
and vacuolating vrrur of monkeys. The family
Papovaviridac has two genera - Potyvmzvirvs
which contains the simian vacuolating virus iSVr
40!■ and polyomaviruses, and PtfpjJfojtiavrrus
coni,Lining hiirn.m and artirliAJ papilloma VuUSes.
They are smallf nonenveioped, Lcosahcdral DNA
tumour viruses. M u st of the naturally occurring
papovavirus run-tours jtl1 benign, but some such as
rabbit papillotna are potent] :iiiy malignant. Polyoma
and the vacuolating virus SV 4f) produce malignant
tumours when inoculated into newborn mice or
hamsters. These viruses have been widely employed
in the study of viial oncogenesis.
Myoma virus often causes latent injection in
laboratory mice. However, when inoculated into
newborn mice, ic produces a wide variety of
malignant rumours. Hence the name ^jolyoma.
The simian vacuolating virus (SV 40) was isolated
from unitwaikred rhesus and cynomolgus monkey
kidney tissue cultures. The yiius did not product any
cytnpaihic effects in the original cultures but when
fluid from such cultures was inoculated into kidney
cell cultures from other simian species (African green
or griver monkey), cyropathic effects occurred,
consignng of prominent cytoplasmic vacuolation. SV
40isoncogenii in newborn hamsters. Its only medical
importance is that because of its oncogenic potential,
live viral vaccines should be manufactured only in
monkey kidney ii$sue cultures tested and found free
from SV 40 infection.
Papillomaviruses are species specitic and infect
squamous epithelia and mucous membranes,
inducing different types of warts or papillomata in
their hosts. Human papillomavirus (HPV) infects
only humans and grows only in organ cultures of
human skin. Over 70 types of HPV have been
recognised based on genetic homology. Them is
correlation between the virus type and the type of
lesion produced. Common warts (verruca vulgaris)
usually found on the hands and feet of children
and adolescents arc mostly caused by types ], 2, d
and 4 r Condyloma acuminatum or genital wart
which is a more moist, soft, pedunculated wart
found on the external genitalia is usually due to
types band 11.This may be transmitted vene really
and may OCCis ion illy turn malignant. There i> a
close association between specific HPV types and
genital malignancies in both sexes. HPV types 6
and 11 are associated with intraepithelial neoplasia,
while HPV 16 and IS arc ecologically related to
more severe invasive malignancies such as uterine
cervical cancer. CnfaCtnr? appear to he important
in the induction of HPV associated malignancies.
Human papovaviruses have been isolated from
a number of patients wuh impaired immunity. The
JC virus was isolated i:i 1971 from the brain of a
patient with Hodgkin's disease and progressive
multifocal leukocmccphalopathy (PML). Isolation
of similar strains from PM L cases has been reported
from the USA* UK and Fr&nce.The JC virus glows
only in human fetal glial cell cultures. It is
oncogenic, producing malignant gliomas following
intracerebral inoculation in newborn hamsters.
Another human papovavirus is the BK virus.
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 ~\‘7 ' r.'L *r

f i ______ V ■

■. , >:
i

C o p y rig h te d m ate rial

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coronavinis' in memory of Dr Carlo Lrbani who
identified tbe new epidemic, initiated early s te p s
for its control and died of it within a month!
The [irmly Rcoviridac derives its name from the
jirotoiypL' virus which wis known as the Respiratory
Lnteric Orphan virus, because it could be isolated
frequently from the respiratory and enteric tracts, but
was not associated with any disease. Members of this
tan dlv are double shelled icosahedm] viiutcs, 5 5 -5 7
Tim in diameter, The genoQVe consists of double
stranded RN A in 1 0 -1 2 pieces, a feature unique
among animal viruses.They are nonenveloptd and
resistant to lipid solvents. The family contains three
genera— Rcovimx, Orbivirut and Rotavirm,
The genus ftewirvs contains Three mammalian
serotypes (Keovirus types l t 2 and 3 j which p<KSess
. iiTui* : 1
a common complement-fixing antigen but can be
dilfefL-n bated. by neutralisation lots. Reoviruses
agglutinate human erythrncytev. Hemagglutination
is type specific and is neutralised by the specific
antibody Rcovtmses have not been proved to cause
any human disease.
k, t ’ -I : -x
'ITiesc have a double shell in which the outer layer
is fuzzy and indistinct. The inner layer has 32 : mg
shaped Lrapsomers. The nime nrhivims is derived
from nrba, in Latin, meaning ring. Orbivi ruses
multiply in insects and vertebrate* thus qualifying
Ai arboviruses. They are responsible for veterinary
diseases such as African horse sickness and blue
tongue, The only known orbivitus infection of
human beings is Colorado tick fever.
'" t n Y / j; :s
These duublc walled viruses present a L'liaraLterislie
appearance under the e le ctro n m icro sco p e.
C o p y rig h te d m aterial
Hidden page
 Rotavirus vactints have been developed* but nre
not in use now.
Reside* rotaviruses, the followi rig viruses are known
or suspected to cause diarrheal disease:
A 21 nm virus was shown to
be responsible for an epidemic of gastroenteritis,
affecting school children and teachers in. Norwalk,
Ohio, in 1972. The virus induced diarrhea in
human volunteers. Serological surveys have
shown that infection with Norwalk vrtuS ]&
widespread in many countries. Extensive epidemics
of Norwalk virus diarrhea associated with
consumption of tiw oysters have been reported from
Australia mul America, It appears to be an important
cause cd diarrhea ill adults ifld children.
The vims can he tlemnnstrared in frees bycjcCtKUX
microscopy, Antibody to the vims car be detected by
immune electron microscopy and radioimmunoissafv:
It has not vet been propagated in cell cultures. Iattic
is Ion >w tj about the yropertws of the virus, 1C.his been
included in rhe family Cshcrvirpciac consisting nf
small round RNA viruses, 22—IQ nm in size, many
of which have been reported from diarrheal feces.
The name caUdvirus is derived from the presence
of 12 cup shaped depressions on the vims surface
(from calyx, meaning cup).
Several outbreaks of diarrhea in
dubfceo have been associated with the presence
of large number?; of adenoviruses in feces. These
adenoviruses can he only grown with difliL-ulty in tissue
culture, 'Jltey have been designated types 4(1 and 41.
Adenovirus aswuciatcd diarrlwea has been seen more
often in summer months.
These scar shaped, 28 nm isometric
particles ha vs been associated with some epidemics
of diarrhea in children. Similar viruses have also
been identified in lamb and calf diarrhea.
These are well established causes
of acute diarrhea in calves, piglets and dogs. They
have been observed in human feces also but their
relation to diarrhea is uncertain.
Two new
zoonotic viruses, closely related hut distinct arc
Hendna (1994) and Nipah (1999} viruses named
after the place they were first isolated in, in
Australia and Malaysia respectively. They have
been classified under the family Fuunyxoviridu.
They are prevalent in Australia and in Malaysia,
Indonesia. Philippines arid the Pacific Inlands.
Their natural hosts are fruit bats in which they
cause silent infection. Symptomatic infection occurs
in horses, pigs and many other domesticated wild
animals. Human infection is usually subclinical, but
may be a flulike fever which may lead to fatal
encephalitis, sometimes as outbreaks. Hendra virus
outbreaks have been minor, but nipah virus has
caused Large outbreaks, with ease facility fates of
up to per cent in persons in contact with pigs.
Large scale slaughter of pigs had been undertaken
in order to stop outbreaks.

Burke K Jivd 11 DcsseLbcrger 19**6. tiucaviruj pathogenesis, Lamer 8:299-

Ciner MJ and WT) Cubiilt 1995. Norwalk and related vinucs. C’urr Oprn Lifter Dii 3:403.
Fields BN' ec iL 1996. Virology, 5“ edn- Philadelphia; l.ipy:ncnr-t-Jl.ivEii.
Johnson KM et al. 198^. Clinical virology of L w ftwr J Jnt'cct IJj's 155:456.
K ap ikian A Z . 1994. V ira l in fe c tio n * p f the gaH roin testijifll tr a c t N e w Y o rk M a ie a l-D e k k c r
Lee HW and ( j! Van der Cnrcn 1939. Hemerrhagic fever with renal syndrome. FrogrMed K ltJ36:62,
Holme* KV. 20U3. SARS. New E n gJ Med 348; 1940.
Frusmcr SR. 199B. Prions of humans and animals. In Topkytnd li'jfiairl-' MkwbioSagJ ?ad Micrabiaf lnfeK-tionjh9lf edn.
London: Arnold.
Richmond JK and DJ Baglole 3003. Lassa Fm r, tfr M edj3tfirt7l-
WeiHfil RA and MB Edmond 2003. Managing bARh. New E n#J Mecf 348:1947.

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RNA VIRUSES
I. 1. Avian leukosis viruses
2. Murine leukosis viruses
3. Murine mammary
Tumour vims
4. Lenkcttii-Siiivoiiia finis
of vauous -animals
5. Human T cell leulccmia
viruses
DMA VIRUSES
I. Papovavinis 1. PapiUumaYiruses of
human beings, rabbits
and ether animals
2. Potyomavirus
3. Simian vims 40
4. B K and JV viruses
IT. F&xviru* 1. MoLkisaim wntagiofum
2. Yaba virus
Shape fibroma
III. Adenovirus Many human and
ni>nhiim»ii types
IV- Htfpeivirut 1. March* disease virus
2. L u c k c 's fro g tu m o u rv iru s
3. Heipes situs pan, papio,
aides and saltruri
4. Epstein-Burr virus
5. Herpes simplex virus
Types 1 and2
6. Cytomegalovirus
V. Hepacitii B and
viruses
(Tal>!r 61.1). Transformation from normal to
malignant cell is a multistep process and may be
partial or complete. For nunple, snme viral scents
can 'immortalise' infected cells, so that they become
capable of continuous multiplication in culture,
without possessing other features of malignancy.
Tnnsfflnnttion is recognised primarily by a change
in morphology of cultured cells. Transformed cells
are altered in shape and lose the property of “contact
inhibition’ so that, instead oi growing as monolayer,
they grow piled up, one over another, forming
“microtumours1. Foci of transformation can easily
be made out and are used in the assay of oncogenic
viruses, such as the Rous sarcoma vims.
About a quarter of the 600 or bo animal viruses
possess oncogenic potential ('Fable 61.2). The
viruses associated with cancers in human beings
are shown in Table 61,3. Both RNA and UNA
viruses are oncogenic. While all oncogenic RNA
viruses (formerly called oncornaviruses) belong to
a single family (retrovirus), oncogen ic viruses occur
among all major groups of DNA viruses,, except
parvovirus. Retmviruses arc responsible for naturally
occurring leukemia and sarcoma in several species
of animals. Among DNA viruses, some
herpesviruses and hepadnavi ruses cause malignant
(utnoLirc in their natural floats.
Papilloma viruses cause benign tumours in their
natural hosts but Some o f them (e.g., condyloma
acuminatum in humans, rabbit papilloma) mav turn
m alig n an t. The a sso c iation between human
papilloma virus (H PV ) infection and cancer of
cervix uteri, particularly HE>Vr types 16 and IS has
been established. The continuous cell line He La.
derived many decades ago from a cervical
carcinoma and used widely in various laboratories,
has been found to contain H PV -lS UNA. In
general, infectious virus particles cannot be
demonstrated in tumours induced by UNA viruses
but papilloma in the wild cotton tail rabbit is an
exception. Rabbit papilloma virus, or DNA
extracted from it, can produce papilloma in rabbit*
following subcutaneous injection.
The polyoma virus causes natural latent
infection in laboratory and domestic mice.
I Jowever, when injected into infant mice or other
rodents. it induces a wide variety of histologically
diverse tumours. The virus can be cultivated in
mouse embryo fibroblasts or baby hamster kidney
cells, in which it induces transformation. The
polyoma virus produces a hemagglutinin.
The papovaviruses BK and JC , which cause
widespread asymptomatic human infection, can
induce tumours in immutiodelkienE subjects.
Simian virus 4 0 (SV 4 0 ) was discovered in
C o p y rig h te d m aterial
 Papovaviridae Human pslpilkulia virus
Heipes-viridie F.-B Virus
H 5Y typ e2
Hepidnaviridne Hepatitis B virus
Flaviviridae Hepatitis C virus
RetrovLridaee H TL vims
apparently normal monkey kidney cultures used far
the producdon of the polio vaccine, It causes an
inapparcnr infection in rhesus and cynomolgus
monkeys and docs not cau.sc cvTupathit: effects in
cell cultures from such monkeys. However, when
fluid From aucli cultures is inoculated inco renal
cell cultures derived from African green monkeys,
cytopathic change with prominent cytoplasmic
vacuolodoo result?. Injection inm newborn hamster?
produces tumours. Trans ton nation is induced in
cultured cells from several species, including human
cells. Millions of doses of polio vaccine prepared
in monkey kidney cultures that may have harboured
SV 40 virus had been used before tbc virus was
discovered. These individuals have been followed
up for over 25 years and no SV 4Q-related tumour?
have been reported. There was considerable
apprehension when [fie oncogenic effect of SV 40
was discovered. However, there ls no evidence that
the injection of vaccine containing SV 40 has
induced cancer in humans.
Three members ut" the poxvirus group induce
benign tumours, rabbit fibroma* molluscum
contagioHiin and Yaba virus. The last causes
it a rurally occurring benign histiocytomas in
monkeys. It is apparently transmitted by insects.
Similar tumours can be induced experimentally in
many specie? of primates, including human beings.
The tumours regress spontaneously in a few weeks,
Nonpri mures are unsusceptible.
Cervical, vulvar, penile cancers
Squamous cell carcinoma
Nasopharyngeal carcinoma
African Burkiti s tymphonu
U cell lymphoma, Cervical cueinoma
Hepatocellular carcinoma
Adult T cell leukemia
Though ionic types- ( I 2 r 1^* 21) of human
adenovirus may produce sarcomas in newborn
rodents after experimental inoculationhthey do not
appear to have any association with human cancer
Many berpesviruses hate been associated with
natural cancers in animals nrid humans.
This is a fatal contagious
ncurolymphomatosis of chickens. No infectious
virus particle can be isolated from the lesions or
seen under the electron microscope. However, sick
hirds shed large quantities of virus from their
feather follicles. The virus is a typical herpesvirus
Maick's disease can be induced in young chicken
hy the injection of the vims. The virus grows well
in chick embryo fihroblasrs producing cyrapithic
changes but no evidence of transformation, MaiekN s
disease can be prevented by a live avirulcnt vaccine.
This Lb tile First instance of a malign .ml disease
being controlled by a viral vaccine.
A herpesvirus
is considered to be the etiological agent of a renal
adenocarcinoma in frogs.
This virus was
isolated from a culture of Squirrel monkey kidney
celts. It causes fetal lymphoma or reticulum cell
sarcoma when injected into owl, monkeys nr
rabbits. Herpesvirus saimiri infection has been
suggested an a primate model for the study of
C o p y rig h te d m ateri
interactions between the EB virus and human
beings.
A herpesvirus, called
the EpStein-Barr virus, is faund regularly in
Cultured lymphotytrt from Burkitts Ivmphoma
patients. In the botlv* the tumour cells contain no
virus but cell lines established from them contain
5 -2 0 per cent of cells that produce the virus. The
virus multiplies only in human lymphoid cell lines.
Serological surveys show char infection. with the
virus is worldwide. Infection is usually
asymptomatic. In you ng adults without prc^existi ng
antibodies, EB virus infection induces infectious
mononucleosis. Lymphoma is believed to occur
when the infection taken place in children whose
immune systems ate compromised, as fer instance,
by chronic malaria, EB virus associated lymphomas
have been reported in transplant recipients. EE1V
has also been linked to nasopharyngeal carcinoma
in the Chinese male population in southeast Asia
and East Africa,
An association has been proposed between herpes
simples type 2 infection and cancer of the uterine
cervix, though not proved. It baa also been suggested:
tb it herpes simplex type 1 infection may be
associated with cancer of the lip. Herpesvirus
type S has been linked to Kaposi’s sarcoma.
infection has been
associated with carcinoma of the prostate and
Kaposi Sarcoma.
HBV has been claimed to be directly or indirectly
involved in the etiology of hepatocellular
carcinom a. Studies in many countries have
demonstrated an enema prevalence ot markers of
H BV infection in patients with primary
hepatocellular carcinoma as compared with
matched controls or with the general population.
Hepatitis C virus infection Jills also b e e n reported
to lea d to hepatocellular carcinoma.
Retroviruses arc enveloped, spherical viruses that
are released by budding through the host cell
membrane. I ’hey are approximately 100 nm in size.
The genome Consists of two identical, linear, single
stranded RNA molecules. The itosahedral
nucleon apsid core encloses the helical
ribonudtoprotein and is surrounded by an envelope
composed of glycoprotein and Lipid,
The characteristic feature of retroviruses is the
presence within the virion of the unusual enzyme
RNA depend ent DNA polymerase or reverse
transcriptase (hence the name nerro, meaning
.reverse). Unlike the classical transcription of generic
information from DNA to RNA, the reverse
transcriptase enzyme prepares a DNA copy of the
retroviral RNA genome— initially an RNArDNA
hybrid and then its double stranded DNA form.
The double stranded DNA form of retroviral
genome, called the provrnjs, is integrated into the
DNA of the infected host ceil. It is from the proviius
[hat all retrovirus proteins are translated- Infection
with oncogenic retroviruses does not lead to
cytolysis or death of infected cells, but die provmis
remains integrated with the host cell DNA for the
rest of the life of the celL
While all oncogenic RNA viruses belong to the
family J?etrovjrjffaes *11 rctrovirustH ate not
oncogenic,The family /icivovjridaf is classified into
three subfamilies.
1. OnC a v i r i n n e comprising all oncogenic RNA
viruses (formerly called oncornavirus).
2. SpUAunhiiae containing the nonnneogenic
foamy viruses' = foam) causing
asymptomatic infection in several animal specie^
and presenting as contaminants of primary cell
cultures in which ihev induce feamy degeneration.
2. Lcnavuinsc including the viruses causing ‘slow
infections' [fentw = slow) in animals (seechapter
60), as well as the human and related animal
immunodeficiency viruses (see chapter 62}.
C o p y rig h te d m aterial
 Rti rovaruses arc widely distri bidted, be ing fbu nd quantifies of interleukin-2 . I'hc closely
in nearly all VcrtebfaTieSj including Uliinalt, birds related HTLV-1I is also j ■ wirh T cell
and reptile*- Rased nn the host range and typos i>f mali]p1iiX JtH T L V ulftctiiiil is ■to be spread
disease caused, oncogenic retroviruses can be through blood transfusion and , methods of
considered under ibc following groups: transfer of leucocytes,
rr .................... ■ . . ; A group ■ . Retroviruses usually infect
of ancLgenically related viruses which induce avian only one host species, the specificif) being
1eli koHi h (Lymphomatosis, myeloblaswiis and ccuiditioned mainly by the t u at i)ce of viral iccepiors
crythrobJasrosis vimscs) or sarcoma in frauds (Rous (]n the host cell surface, i ■-. rdii . oil their ability
sarcoma virus, RSVJ, to grow In ocEls from different . .retroviruses
Tills group have been classified into [1) erotrupi (multiplying
consists o f ^vera] strains of murine leukemia and in cells of native Kcsnt species i:! - ■. 1. miphotmpu
sarcoma viruses,. named after the investigators who (multiplying in cells of native u I species);
first described them (e,g. Gross, Kricnd, Moloney, and (3) xenotropic (m hiptylng mi* in ceils of
Rauscher). foreign species but noi of native huci species).
r ■- r J! -I lrT aB 1
■ T ins Two rypes o f retrovirus
virus occurs in certain strains of mice having a high transmission occur. Exogenous errov]rust are
natural incidence of breast cancer. It used to be spread horizontally, Most oncogeni retroviruses
known as the Jmi3k factor' or ‘Bittner virus'. It ■ire exogen ous. End ogen ou s rr r. are
multiplies in the mammary gland and is transmitted transmitted vertically from parvnr to offspring, by
■ frum mother to off-spririg t!irough breast milk. Mice the provirus integrated with le germ line cell
can be infected by oral, subcutaneous or genome. The endogenous retrovirus provirus
iritrj]ierL(oneaJ mutes. Mammary CflO(£r occurs unly behaves tike a cellular gene. id is subject to
in susceptible strains of mice, after a Latent ]ieri(jd
of 6 ~ 1 2 months,
i_ i , "L
■ A number ot viruses have been isolated
from leukosis and sarcomas in various species of
animals - cat, hamster, rat, guinea pig and monkey.
r“" -■ ; f‘ ; *"] f,.7 ' rr;

Retroviruses named human rf cell leukemia viruses

were isolated in 1980 from cell cultures from adult
patients with cutaneous T cell lymphoma (mycosis
fungoidcs) and leukemia (Senary syndrome) in fbe
USA. Similar viruses have beer isolated from
patients with adutr T cell leukemia in japan and
the Caribbean. HTLV rype 1 is present worldwide
but the disease is limired to endemic areas. Besides
adult T cell leukemia, HTLV-1 has also been
associated with tropical spastic paraparesis,, a
demyelinating disease. The vims preferentially
infeeisT4 (CD4) cells. Infected T cells express large

C o p y rig h te d m a te ria l
 LTR
gog
U LTR
JfOJC pal
c LTk
Jpaje
A- Basin- ictn?vin,i? Aviwi Iwktntia lin K ^ tlw n illfe in iA g 'nrvs?*
B. TranHeguUting; retroviruses-e.g. HTLV, HJV
C. Acute cransfornim^ rftnovjnit'i onooyciH: replacing part ofbasic genome, RepLic^cior defective,
regulatory t o r fro! by the host cell. Endogenous
retroviruses. are usually silent and [Jo not transform
cells or cause any disease, llic y can he detected
either by activation alter exposure to radiation or
chemicals, or by nucleic acid hybridisation
techniques.
Retroviruses are labile, being
inactivated at 56 uU in 30 minures, by mild acids,
ether and formalin. They are stable at -3 0 *C.
Two types of antigens are present—
the type specific glycoprotein antigens on the
envelope, and the group specific nuclcopnotcin
antigens in the virion core. Cross-reactions do not
occu r between surface antigens of retroviruses from,
different host species.
Retroviruses have a
relativety simple genomic structure (Fig. 61.2).
The proirinis of a standard retrovirus (such as a
nondefectivc avian or murine leukemia virus)
consists of three genes required for viral replication
- gag, poi, and env in that order from the 5' to T
end. The gag gpne codes for the nueleocapsid core
proteins which are group specific antigens (hence
LTH
p(*i MV
LTR
env mi
LTR
, 'Ml
the name gag); the poi gene encodes the RNA
dependent DNA polymerase; and the env gene
encodes the envelope glycoproteins. Long terminal
repeat (LTR) sequences are present at cither end
of the provirus and linked directly to the host DNA.
The LTRs exert regulatory control on the provirus
gene functions.
Some retroviruses (transregulating viruses) such
as HTLV or HIV carry a fourth gene, ase or Mt,
after the env gene. This is a tran-KBCtivuting gene
that regulates the Junction of viraE genes.
The standard oncogenic retroviruses, such as
chronic leukemia viruses, are slow transforming
viruses, i.e., they have a low nnco^enic potential
and Induce malignant change, generally only of
blood cells., after a long latent period. They do not
transform cultured cells. They are capable of
replicating normally. In contrast, rfre acute
tronsfcmuqgvfnjses arc highly oncogenic and cause
malignancy after a short latent period of weeks, or
months. They can cause different types of
malignancies - sarcoma, carcinoma, leukemia-and
also transform cells in culture. However, rtiO&C acute
C o p y rig h te d m aterial
580 * Textbook oF Microbiology ►
transforming viruses are unable to replicate
normally because they carry on their ^ ro n it an
additional gene, the viral oncogene {V-onc gene)
which replaces some of the genes essential for viral
replication. Such Y^onc viruses can replicate only
if co-infected with a standard helper retrovirus.
The Rous sarcoma virus which carries the
oncogenic arc (pronounced 'Sort1) is the best known
among acute transforming Viruses but it is different
in being jTc^Jri-afrrjn competent, that is. it can
replicate normally because it posscses the full
complement of gig, poi and m r genomes. Mosc
acute transforming viruses arc replication defective,
ONCOGENES
Viral oncogenes (V-onc), commonly known as
'cancer gene}-' arc genes which encode proteins
triggering transformation of normal cells into cancer
cells. Oncogenes arc not csscr ti al for the repli catiion
of the vims and mutants lacking them occur, which
replicate normally without being oncogen ic.
Genes closely resembling viral oncogenes are
found in normal as well as cancer cells. Oncogenes
isolated from cancer cells arc called cellular
oncogenes fC-onc). Similar genes found in normal
cells are called pro torn neocenes They are not of
vital origin. On the contrary viral oncogenes appear
no be of host cell origin. Cellular oncogenes contain
intronE characterise c of eukaryotic genes, whereas
viral oncogenes do not. Apparently viral oncogenes
originated at some distant past from proto-
oncogcnes by recombination between retroviral and
cdlular genes.
Proto-oncogenes arc widespread in vertebrates
and metazoa—from human beings to fruitflies.
They ate well conserved in their genomes,
suggesting that they serve some essential functions
in normal cells. They have been found to code for
proteins involved in regulating cell growth and
differentiation. The presumed functions of many
oncogenes have been identified. For example, the
oncogene srr i> related to tryiosine-speuific protein
kinases, sis no a platelet derived growth factor, and
myc to DNA-binding proteins, all concerned with
regulation o f norma] cell growth..
A useful method for the study of oncogenes is
transfection. Certain mouse fibroblastccD lines, such
as N1H j T3, can rake up foreign DN A, incorporate
them into their genome and express tnmsfecTJonr
By this technique, DNA extracted from human
rumour cells has been shown to transform 3T3
cells, and such transforming genes have been shown
to be identical with cellular oncogenes.
A n t [-G n c o q b n b s
A class of genes has been identified in normal
retinoblastoma (Rb) gene, the loss of which is
associated with the development of retinoblastoma
in children. The p53 gene appears to be a tumour
suppressor gene with a wide range of effects.
Specific chromosomal deletions, recognised in
association with certain types of human cancers may
reflect the loss of tumour suppressor genes.
M ec h a n ism s of V ir a l O ncoornbsis
While it is known that oncogenic viruses are able
to transform cells in culture and induce tumours in
animals, under natural or experimental conditions,
the exact mechanisms of viral oncogenes:* are not
well understood. Malignancy is a stable heritable
change and, as such, should be the result of a
modi treat ion of the host cell genome.
In the case of oncogenic DNA viruses, the viral
DNA (or a portion of n} is integrated with the
host cell genome. The viral DNA being incomplete
Or 'defective1, no infectious virus Is produced.
However, under its i itfluence, the host cell undergoes
malignant transformation. A virus transformed
cancer cell is in many ways analogous to a
bacterium lysogeruerd by a defective phage. In both
cases, the cell is not destroyed and no virus is
produced. Acquisition of new characteristics by the
transformed cell resembles lysogenic conversion in
bacteria.
In general, retroviruses induce tumours by two
mechanisms— either by introducing into the
 V-STS chicken &wwma C-src 20
V-rw rat Sarcoma C-rw 11
V-myc chicken Leukemia C-myc 3
V-fa cat Shwiaji c -r« 15
V-sis monkey Sarcoma C-sis 22
V-mos mouie Sarcoma C-mos 3
1 Oncogens are p'-'en three Luttcr coirs from the animal or tumour from which they are derived, preceded fry
cither V- or C-, for viral or cellular gens respectively;
sre ■ sarcoma of chicken, ras - rdt sarcoma, ns - simian sarcoma,

cellular genome a new transforming jrenc
(oncogene) or by inducing or alteri ng the expression
of a pre-existing cellular gene. Several molecular
mechanisms have beer suggested for the conversion
of benign proto-oncogenes to cancer genes. The
genes truly get overexpres'sed and the overproduced
gene product may lead to abnormal growth.
Recombination between retroviral and. cellular
genes, promoter insertion, chromosomal
translocation, gene amplification and mutation are
some of the genetic processes relevant in this
connection.

LinlnJpJM. 19S9 I"hr iiiukculuj yruLLu's u( cjulli. Siimix: 235JOS.

Friendf SH ef aJ. 1900. Oncogenes and tumour suppressor genes. New Eng! j Aind 318:618.
NcjI MS and _[AWykc 1998, Viral oncogenicity In JfrpJ^f and Wiltons MkTvbioiqgy and JVfjcrobj'ai Infections, 9lhedn.
Vfrl.l. LondomAmuld,
V*rmus H, 1988- Retroviruses. ScrencT 1427-
Vi'rinfreTir RA, 1983. Finding tht antiotlCqKeiK-, Snirnr Anw 259:44.
VfejnbcipRA 1989, Oncogenes, antiuciccigenes and the molecular basis of carcinogene^s, Canerr fie-v 49^3713.

C o p y rig h te d m aterial
Hidden page
in 2003r Three million patients died of AIDS in
2003.
HIMANiflUioOEKlGEfCY VIRUSQW)
HIV, the etiological agent of AIDS, belongs to the
lentivirus subgroup oi the family ReUoviridae.The
lenthrirus subgroup includes the causative agents
uf the slow virus diseases visna/maedi in sheep and
infectious anaemia in goats and horses. Besides
HIV, the related animal immunodeficiency viruses
also arc assigned Do this group (Table 62.1).
11IV is a spherical enveloped virus, about 90 120
nm in siae (Fig. 62.1). The nucleocapsid has a.n
outer icusahedrul shell and an inner coneshapcd
core, enclosing rhe ritwnucleoprcrteins.The genome
is diploid, composed of two identical single
stranded,positive sense RNAcopics. In association
with viral RNA is the reverse Transcriptase enzyme,
which is i characteristic feature of retroviruses.
When the virus infects a cell!, the viral RNA is
transcribed by the enavme, first into single stranded
DNA and then to double stranded DNA (provims)
which is integrated into the host cell chromosome.
The proving can remain latent for long periods,
though it influences host cell functions. At times,
in response to viral promoters, the provims initiates
viral replication by directing synthesis of viral RNA
and orher components
During viral replication, when the naked virus
in ills out through the host cell surface membrane!
it acquires a lipoprotein envelope, which consists
of lipid derived from the host cell membrane and
glycoproteins which are vinqs coded. The major
virus coded envelope proteins are the projecting
knoblike spikes on the surface and the anchoring
transmembrane pedicles.'l'hc spikes, constitute the
major surface component of the vims winch hinds
to the CD4 receptors on susceptible host cells.
Transmembrane pedicles cause cell fusion.
V i r a l G b n b s a w is* A n t i g e n s
The genome of H IV contains the three structural
genes (gag, pol and env) characteristic of all
netrovi roses, as well as other nonstructural and
regulatory genes specific for the virus {Fig- 62,2).
The products of these genes, both structural and
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Hidden page
 4 Hyman Immungdeficiency Virus: AIDS ►
prevalent ail over the work! belong to HJV type 1.
HIV strains, first isolated from West Africa in 1986t
which react with HIV type 1 antiserum very weakly
or not at ill have been termed HIV type 2. The
envelope a u tism of the two types are different,
though their core polypeptides. chow seme crocs
reactivity. H1V-2 has only 40 per cent genetic
identity with HIV-1. It is more closely related to
simian immunodeficiency virus than to HIV-1. It
is much less virulent than H IV -1. It is largely
confined to West Africa, though isolations have
been reported from some other areas, including
western and southern India.
HIV-1 strains have been classified into at least
ten subtypes based on sequence analysis of their
gag and env genes. These subtypes arc designated
as A to J and constitute Gn>u]i M (for ‘rniior'),
which cause the large majority of HJV-] infections
worldwide. A few HIV-1 strains isolated from West
Africa (Cameroon, Gabon) do not fall within the
Group M and have been designated Group O (for
'(jutlier'), Some later isolates of H IV -1 from
Cameroon, distinct from M and O groups have been
called Group N (for new).
H IV -1 subtypes chow a geographical
distribution, though this is often blurred by viral
trafficking- All known HIV virus groups and
subtypes are present in Cameroon, West Africa,
which may perhaps be the site of origin of the
virus. Subtype A is the most prevalent, being found
worldwide, while B is the most common in the
Americas and Europe. The common subtypes in
Afnca are A, C and D f while in Asia the common
subtypes ate E h C and B. Subtype E Ls ihe
commonest in Thailand. In India and China,
subtype C is the most prevalent.
Antigenic differences between HIV strains may
be Important in gerodiagposis. Infection by HIV-1
or 2 may not be identified unless the corresponding
type is represented in the test antigen. Even with
H IV -1 strains, the differences between the
subgroups are significant enough so that Group O
■m rains may be missed if the test antigen contains
58S
Group M only. Even subtype differences may cause
discrepancies so it may be advantageous to use
antigens containing the prevalent subtypes in
different countries.
The subtypes seem to vary in frequency of
transmissibiliTy by different routes. The suhtypes
common in Asia and Africa fC and E) are more
readily transm iefied by heterosexual contact than the
American strains (subtype B) which are
preferentially spread through blood - by imention
and homosexual contact (homosexual transmission
is considered to be bloodbotne as the virus is likely
tn enter directly into the blood through minor
mucusal lean).
Differences in grow th c h a ra c te ris tic s ire
sometimes observed between HIV isolates from
asymptomatic carriers and from AIDS patients,The
former grow slowly and infect only the peripheral
blood lymphocytes, while the latter grow foster and
yield high litres in established cell lines of
lymphoid and monocyioid origin. Strain variations
may account for differences in clinical course of
HIV infected persons,
R e s is ta n c e : H IV is thermo labile, being
inactivated in 10 minutes at 60 "C and in seconds
at 100 °C, At room temperature (20-25 GC), in
dried blood it may survive for upto seven days. A,
At autopsy, HIV has been isolated from various
tissues upto 16 days after death.lt withstands
lyophillation. The virus in lyophilised blood
products can be inactivated by heating at 68 °C for
72 hours and in liquid plasma at60 GC for 10 hours.
Tabic 622 Major antigens ol HIV
A Envelope antigens:
1. Spike antigen - gpl2Q
(Principal envelope antigen)
2 Transmembrane pedicle protein -’gp 41
B. Shell antigen
J r Nucteocapsid protein - plS
C. Core antigens:
1. Principal core antigen. — p24
2. Other core antigens - pl5, p55
D. PolyrnerHM: antigens — p31, p51, p66
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Hidden page
HLV can be isolated from the blood, lymphocytes,
cell - Lite- plasma, senien, cervical secretions, saliva*
tears, urine and breast milk,
The primary pathogenic mechanism in HIV
infection is the damage caused to the CD4+ T
lymphocyte, The T 4 cells decrease in numbers and
thcT4:T8 (helper: suppressor) cell ratio is reversed.
Viral infection can suppress the function of infected
cells without causing structural damage. Injected
T 4 cells do not appear to release normal amounts
of interleukin^, gamma interferon and other
lymphoid nes. Thin has ft marked damping effect
cm cell mediated immune responsc.
Thougb the m ajor damage ;J to cellular
immunity, humoral mechanisms also are affected.
Helper T cell activity is essential for optimal B cell
function, particularly in responding to thvmus
dependent antigens. AIDS patier-fs are unahk to
respond to new antigens. An important feature in
1IIV infection is the polyclonal activation of B
lymphocytes leading ro hypergammaglobulinemia.
AH classes of immunoglobulins are involved hut
levels of IgG and IgA are particularly raised. In
infants and children, IgM levels also are elevated.
The hypergammaglobulinemia is more a hindrance
than a help because it ls composed majulvof useless-
immunoglobulin to irrelevant antigens and also
autoantibodies, This may also be responsible for
allergic reactions due to immune completes (type
3 hypersensitivity).
Monocyte 'macrophage funotion is also affected
apparently due to lack of secretion of activating
factors by [lie T 4 lymphocytes. As a result,
chemotaxis, antigen presentation and intracellular
killing by monocytes/macrophages are diminished.
The activity of NK. cells and cytotoxic T
lymphocytes is also affected. The principal
immunological abnormalities seen in HIV infection
are listed in Tabic 62.3.
Qinical manifestations in HIV infections are
due not primarily to viral cytopathology but arc
secondary to the failure of immune responses-This
renders the patient susceptible to opportunistic
infections and malignancies. An exception to this
may be the dementia and other degenerative
neurological lesions seen in AIDS, These may be
due to the direct effect of HIV on the central
nervous system.
AIDS is only the last stage in the wide spectTum of
clinical features in HIV infection. The Centers tor
Disease Control (USA) have classified the clinical
course of HIV infection under various groups (Tabic
62.4).
The natural evolution of HIV infection can be
considered in the following stages:
Within 3 -6 weeks
of infection with I IlV, about 50 per cent of persons
experience low grade fever, malaise, headache,
lymphadenoparhy, sometimes with rash and
arthropathy resembling glandular fever. Rarely,
there may be acute encephalopathy, Spontaneous
resolution occurs within weeks. Tests for HIV
antibodies are usually negative at the onset of the
illness but become positive during its course. Hence
this syndrome has been called 'seroconversion
illness*, though in many of those infected , ‘acute
retroviral syndrome’ or seroconversion occurs
without any apparent illness. HIV antigenemia (p24
antigen) can be demonstrated at the beginning of
this phase. The pathogenesis of seroconversion
illness is believed to be due to immune complexes
as well as to the direct effects of viral multiplication.
All
persona infected with HIV, whether nr not they
experience seroconversion illness, pass through a
phase of symptu miens infection (clinical latency)
which may last up to several yeiJrs. They show
positive HIV antibody tests during this phase and
arc infectious. The infection progresses in course
of time through various stages, C D 4
lymphocytopenia, minor opportunistic infections,
C o p y rig h te d m aterial
persistent generalised lymph adenopathy; A1D5-
rektcd complex (ARC), ultimately terminating in
full blown AIDS, with it? characteristic infections
and malignancies. The median rime between
primary HIV infection and development of AIDS
lms been stated as approximately 10 years. About
5 -1 0 per Lent of llie infected appear to escape
cli nical A IDS for 15 years or more. They have been
termed 'long term survivors’ or 'lung term
nonpm grefsnrs12345. The mechanisms for such
prolonged survival are not dear, though many viral
and hnst determinant^ may he responsible.
This period of clinical latency however dues
nut mean microbiulogical latency as virus
multiplication goes on throughout. "I'he host mounts
an immune response against the virus, both
hu moral and cellular, wn ich can only lim it the virus
load, but not clear it completely. A chronic persistent
infection with varying degrees of viral multiplication
is the result. The CD4+ T cell count decreases
steadily, from over 1000 per microlitre to about
500 or less by the stage of acute infection. When
the count falls to or less, clinical AIDS usually
sets in. For this reason the case definition by GDC
includes all HIV infected cases with CD4* T cell
courts of J0Q or less, irrespective of their dinical
condition.
This has been defined as the
presence of enlarged lymph nodes, at least 1cm, in
diameter, in two or more noncontiguous
entrainguinal sites, that persist for at least three
month*, in the absence of any current illness or
medication that may cause lymphadenopathy. This
by itself is benign but the cases may progress to
ARC or AIDS,
This
group includes patients with considerable
immunodeficiency, suffering from various
constitutional symptom? or minor opportunistic
infectiOnS-The typical constitutional symptoms are
fatigue, unexplained, fever, persistent diarrhea ant]
marked weight loss of more than 10 per cent of
hndy weight.The common opportunistic infections
arc oral candidiasis, herpes zoster, hairy cell
ieueoplakia, salmonellosis or tuberculosis.
Generalised lymph adenopathy and splenomegaly
are usually present. ARC patients are usually
severely ill and many of them progress to AIDS in
a few month*.
This i.s the end-stage disease
representing the irreversible breakdown of immune
defence mechanisms, leaving the patient prey to
progressive opportunistic infections and
malignancies.
The clinical severity of AIDS varies with the
type of infection or malignancy present. In early
AIDS, many patients are ill only during episodes
of infection, which may respond to treatment.
Between episodes they may be relatively well and
able to resume normal life. Patients with Kaposi's
I. Features that characterise AIDS
1. Lymphopenia.
2. Selective T cell defidency-
ReduiTion :n number nf"T4 (CD4)
cells, Inversion ofT4:Tfl rario.
5. Decreased delayed hypcrsenimvity
on skin testing.
4. Hypergammaglobulinemia -
predominantly Igp and IgA;
and IgM aLso in children.
5. Polyclonal acrlvarion of B L-dlx and
increased tfxmtMKdut secretion
oflg.
II. Other consistently observed features:
1. Decreased in vitro lymphocyte
yrolifeiacive response to mitogens
and antigens.
2. Decreased cvtothkil' responses by
T cells and N K cells.
3. Decreased antibody response tn
new antigens.
4. AEtered monocytcAnacmphagC
function.
5. Elevated levels of immune
templates in serum.
Copyrighted material
Hidden page
pathy leading to loss of higher functions,
progressing to dementia.
About a third to half the
number of babies bom ro infected mothers arc
infected with HIV. Virus transmission may occur
to rhe fetus in pregnancy as early as the tirst
trimester, but infection in more common perinatal ly.
Many of the infected children may not survive for
a tear. Children may also acquire the infection from
blood transfusion or blood products.
There are many differences between adult and
pediatric A ID S, Children develop humoral
immunodeficiency early, leading to recurrent
bacterial infections. Failure to thrive, chronic
diarrhea, lymph adenopathy, tuberculosis and
opportunistic bacterial infections arc common
manifeRlatiuns in pediatric AIDS. Lvmphocvtic
irtenitita] pneumonia in seen mostly in children,
while Kaposi sarcoma, toxoplasmosis and
Cryptococcosis are less common than in adults.
Laboratory procedures for the diagnosis of HIV
infection include tests for immuntdeficiency as well
as specific tests for HIV.
The following
parameter* help to establish the immunodeficiency
in HIV infection.
1. Total leucocyte and lymphocyte count to
demonstrate leucopenia and a lymphocyte count
usually below 2000/mm\
2. T cell subset assays. Absolute CD4* T cell count
will he usually iess than SCKVmnf - T4:TB cell
ratio is reversed.
3. Flatekr count will show thrombocytopenia,
J. Raised lgG and IgA levels.
5. Diminished CMI as indicated by skin rests.
6 . Lym ph node biopsy show ing profound
ah normali tics-
T h « e include demonstration of HIV, its antigens
or other components and antibodies and isolation
of the vims.
Following a single massive
infection, as by blood transfusion, the virus
antigens may be detectable in blood after about
two weeks, Jfhc major cone antigen p24 is the
earliest virus marker to appear in blood and is
the one tested for. IgM antibodies appear in
about 4 - 6 weeks, to he followed by lgG
antibodies (Fig, 62.3: Table 62.6).
If the infecting Jose if small, as following a
needle-stick injury, the process may be
considerably delayed. The appearance of p24
untigenemii and vi remil, followed bv IgM
antibody response coincides with the acute or
seroconversion ] 11ness. Afterwards, free p24
antigen disappears from circulation and remains
absent during the long asymptomatic phase, to
reappear onlv when Revere clinical disease sets
in. However anti body-bound p24 antigen
continues to be demonstrable, after dissociation.
The p24 antigen capture assay (F,L1SA) which
uses anri-p24 antibody as the solid phase can be
used for tins. The test is positive in about 30 per
cent of HIV infected persons. W ith prior
dissociation of the antigen-antibody complex,
the positive rate increases to about 50 per cent.
In [he first Few weeks after infection and in the
terminal phase, the test is uniformly positive. The
test is most useful in persons recently exposed
to risk of infection, in whom antibody test is
negative. It is currently used for screening blood
donors in the USA, along with HIV ELISA.
Cnee infected wi rh HIV, a person
remains infected for life. The virus is present in
circulation and body fluids, within lymphocytes
or cell-free. Virus titres parallel p24 times, being
lug]i soon after infection, low and antibody-
bound during the asymptomatic period, and
again high Inwards the end. An infected person
may therefore be infectious throughout, the
infeetivicy being highest in the early phase and
again when the perron becomes terminally ill.
The virus is present in many parts of the body
and can be isolated from the peripheral
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Hidden page
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 K il

have spread to Europe from America, as well is of mitochondrial D N A from human and
directly from the former African colonics of the chimpanzee viruses. It shows that the progenitor
European nations. of H lV -l entered the human population from
Conducive evidence tins now been obtained chimpanzees of the Itibspccief JJjn fru^ioJi Jes
from molecular studies, including genetic typing troglodytes living in equatorial west Africa
(Cameroon, Gabon, equatorial Guinea), where the
virus is considered, on oilier evidence, io have
emerged in humans. HIV's are believed to have been
present in monkeys for over l 0 0 f00Q years.
Transmis&Lon to chimpanzees is a far more recent
ki ■^15 ^ r! il ^r- h f- if* -JO b ptm^ wv’i^ t,
mix b i^h^rr-i-T-afflap iij ii ar^ aj phenomenon and may have hap|jcned through their
■ O* MV» "5
killing and eating monkeys. Human infection could
Sf3 have come from chimpanzees who w t« hunted by
Sr II t p
them and killed for meat. The qpz S1V may have
liken root in humans by becoming HIV through
rt mutation or recombination.
fll
n *i - ;
P

pjr 90 160
M)120
pi

-h* - pea
p5&p 5i .
flLLSul irwu-BT
DP*1
!

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HIV-2

>■s * <«

- * r . *

py ijiieria
 The three groups ot HIV-1 (M, 0 , N) Dfliy women. However, the situation is very different in
rep resen t ind ep en d en t Eran? miss ions from Africa and Asia where men and women are equally
chimpanzees to humans. The source of HIV-2 has affected- In some places, more women arc (bund
already been established as SIV from the sooty infected due to the high rate of infection in
mangabey monkey Ctrcoccbus a tvs. pmstimtes. Transmission in the developing countries
The virus has spread virtually all over rhe world, LSalmost always heterosexual and can take place in
though the prevalence rates in different countries ho|h directions-
vary widely. Initially North America, Brazil, The best method of checking sexual
Western Europe, Austral ia, Central and West Africa transmission (if infection is health education
had high prevalence, while Eastern Europe and regarding the danger of promiscuity and other High
Asia, were only sparsely affected. However this risk activities. Some changes in Hie style and sexual
soon changed. By vigorous measures and active attitudes have already taken place in the USA and
public participation, the developed countries have the incidence in homosexuals has come down.
succeeded in reducing spread of rhe infection, But Persons Indulging in high risk sexual practices and
in many countries of Africa and Asia, the .infecnon spouses of infected persons should be counselled
has spread unhindered reaching epidemic regarding safer sex' methods. The use of condoms
proportions. Differences also exist, both in the offers considerable, though not complete,
modes of infection and in clinical manifestations, protection. The risk of HIV transmission increases
between the affluent and developing countries. Tile with multiple partners.
epidemiology of AIDS has beer studied mostly in
the developed nations and only sketchily in the third
■world, HIV is spread only by three modes— sexual
contact with infected persons (heterosexual or
homosexual): by blood and blood products; and
from infected mo rhe r to babies (intrapartum,
I Sexual intercourse: 0 .1-1.0%
perinatal, postnatal}. There is no evidence of HIV
anal,vaginal, oral
transmission by other means including casual
I] Blood and blood ^90%
contact or through insects. The modes ot products, Factor VTI
transmission of HIV and their relative risks are etc Blood transfusion
shown in Table 62.7. III Tissue and organ 5Q-90fc
HIV is primarily a sexually transmitted infection. donation: semen, cornea,
bone ittjmvw, kidney etc
In the USA it was transmitted predominantly among
IV Injections and 0.5-1 .<JH
male homosexuals. The danger of infection is more injuries: shared needles
for the passive partner because mucosal tears are by drug addicts
very frequent during anal intercourse anti virus laden Injections with unsterile
lymphocytes in the semen can directly enter through syringes and needles
these. One reason for the high incidence of HIV Needle-sock and other
injuries in health -[j.;T ?
infection in male homosexuals may be the large
Surgical wounds
numbers of sexual partners they are reported to V Mother to baby; 30^6
have. In the affluent countries, homosexual and Transplacental
bisexual men are infected far more often than rhe At birth
heterosexuals. For this reason, infection was found After birth
Breast milk
predominantly in men snd only occasionally in

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S^B i Te^rtiook od Microbiology ►

5 m illion H IV -in f e c t e d people, the second largest
such population after South Africa,
Prttplvv I l im s ; T h e prevention o f A I D S rests at
p resent o n g e n e ral m e asu re s s u c h as h e a lth
education, identification o f sources and elim ination
o f h ig h risk a ct]title s . N o s p e c ific vaccin e is
available. T h e h i^ h m utability, diverge antigenic
types and subtypes, long latency and persistence in
infected cells an provirus pose serious problems in
the developm ent o f vaccines. A n ideal vaccine
should not only prevent Infection hut also have
th e ra p e u tic a p p lic a tio n in a s y m p to m a tic
■ neropositives.
T h e lack o f a satiable experimental anim al is a
severe co n strain t. C h im p a n z e e s, m onkeys and
rabbits c a r be infected but they do not develop the
disease. Infection w ith the sim ian immunodeficiency
v iru s is a co n v e n ie n t m o del. Several p o ssib le
strategies have been explored far vaccine production.
T h e se include im m unisatio n w ith fa] m o dified
w ho le v iru s; (h i su b u n its, based on envelope
glycoproteins expressed in anim al cells, bacteria,
viruses ~ -nr as sy n th e tic epitope* on ad juvan t
carriers; and (c) target cell protection by a n t i-C D 4
antibody or genet ically engi neered C 1)4. A number
o f candhlatc vaccines arc being tested in clinical
tri.ds in humans.
F r e n t n ie n c A p p ro a c h e s to the treatm ent o f
A I D S include: (1) the treatment and prophylaxis
o f infecnariH and tumours; (2) general management;
(3) immunorcstor&ttie measures; and (4) specific
a n t :-H T Y agents.
Prom pt diagnosis and approp^uite treatment o f
opportunistic Infections and tumours in the early
stage o f A I D S can be very useful and the patient
m av be able to resum e n o rm a l life in between
episodes o f illness. G e n e ral m anagem ent o f the
patient requires the understanding and oouperation
o f the health slafil in the hospital and o f relatives at
home. G roundless fears about im aginary risks have
to he allayed and reassurance f;iver that the patient
can be kept at home or treated in the hospital
without danger h> contacts, if proper precautions
are taken.
Steps at im m u ro rcsto rativ c therapy such as
adm inistratio n o f in te rle u k in -2 , thym ic factors,
le u c o c y te tra n s fu s io n a n d b o n e m arro w
transplantation have not been very helpful.
Specific treatment w ith antiretroviral drugs is
the mainstay in the management o f H I V infection.
A number o f effective diugs have become available
in recent yean. These include nucleoside analogues
lik e Z id o v u d in e ( A z id o t h m id i tie, A Z T ),
D idanotine, Zalritakinc, Lam ivu d in c and protease
in h ib ito rs like Saquinavir, R ito n a v ir, In d in a v ir,
w hich have been used as monotherapy or in various
co m b in atio n s, A dverse reactions and h ig h cost
restrict their wide use in poor countries.

f u r t h e r Itiidiiii;
ftmder SC *in!TC M?iii£an fed}. 1993. Icrrtxmfc vfAJDS Afadrrfne, London; Williams and Wilkins.
Vim.-. AS HC l-*nc 199R, HW [n HairistNJf F n n d p k t o f In te rn a l Medicine, l4lfl cdtL VoL 2. New York:
M cGraw -Hill.
Centers far Di-tase and PvrvmiiLB), 1993. cla--tifiniciro system for HIV infection Morbidity Mortality
Weekly Report. 41 (RR-17).

C o p y rig h te d m aterial
 Normal Microbial Flora of the Human Body
H u m a n beings [ike other anim als, harbour a wide
j t t .lv o t microorganisms both on and in l I ll- it bodies.
T h e normal microbial flora are mom or less constant
fo r each species and arc b ro ad ly d iv id e d into
rci-idents and transients. T h e former constitute a
constant population w hich cannot be completely
removed permanently* while the larrcr vary from
tim e co tim e and are tem porary, T h e residents
prevent perm anent co lo n isa lu v i o f the body by
other organisms. A knowledge of the normal flora
o f rhe body is essential to an understanding o f the
interaction o f hum an beings and then pathogen
laden environment. Jh.c normal m icrobial flora play
an important role in body economy, T h e y can
(1) become pathogenic when host defences fairer*
(2) prevent or interfere w ith cotonisation/m vasion
o f rhe body by pathogens, (.1} raise the overall
im m une status o f the lurst against pathogens having
related or shared antigens* and (4) cause confusion,
in diagnosis due to their ubiquitous presence in
the body and their resemblance to som e o f rhe
pathogens-. M em bers o f the normal flora foim part
and parcel o f the host and include saprophytes*
c o m m e n s a ls , fa c u lta tiv e p a th o g e n s and true
pathogens.
T h e mieroftora o f die intestinal tract synthesise
vitam in K and several B vi tarn i ns w hich supply on
occasion the body’s needs.T h e antibiotic substances
produced by som e, for exam ple, eolicirts, have a
h arm fu l effect on pathogens. T h e endotoxims
liberated by them may help the defence m echanism
o f the b o d y by trig g e r in g the a lte rn a tiv e
co ni pie merit pathw ay, as lo n g as they arc not
produced in excessive amounts.
O n the contrary, the opportunistic pathogens
am ong them caUi£ disease when the bodys Jetenue
mechanisms fail. T h e ir abnormal m ultiplication can
cause diseases such as enteritis and endotoxic shock,
E^tuidllinase producing organism s L_an aggravate
in fe ctio n by in te rfe rin g w ith therapy* C e rta in
streptococci o f the m outh cause dental caries.
In e n viro n m e n ts laden w ith pathogens for
example, hospitals, a shift in the normal flora o f
the in d iv id u a ls there can cause an increase in
carriage o f antibiotic resistant staphylococci* It has
a ls o been sh o w n th at s u c h p eo p le can he
Tecolnniscd wirh penicillin sensitive staphylococci
o f strain 5 0 2 A w h ic h arc h arm le ss and thus
overcome the damage done, W h e n large number?
o f people congregate from different parts o f the
c o u n try as in a rm y c a m p s , cite new re cru its
experience increased colonisation rates o f Neiss&ia
merjrnirj'ri'df.^ and G r o u p A Strepto co ccus and
viruses su ch as rh in o v jru se s and adenoviruses
sometimes resulting in epidemics,,
T h e hum an skin is constantly and LOntintioUsly
b o m b a rd e d by o rg a n is m s p re se n t in the
environment. It is also contaminated by one's own
secretions and excretions, the extent depending on
the individual's personal hygiene. T h e flora depend
on rhe area o f the body, the clothing one wears,
one's o ccu p atio n and e n v iro n m e n t. T ra n s ie n t
microflora tend to occur more frequently on the
skin*
C u lt u r e s fro m th e sk in have fre q u e n tly
d e m o n stra te d d ip h th e r o id s ( in c lu d in g
C o p y rig h te d m aterial
p rop] u 111 h a e t e r ii) , S tap h y lo co cci (aero b ic and
anaerobic); G ru m positive aerobic spore bearing
bacilli; Srr. \iridanf, 3tr. faocalis-, G ra m negative
bacilli such as E . coir, ProtCUS, nnd other intestinal
o r g a n is m ^ m in i ic u c ; m y c o b a c te ria (n o n -
pathiogicnk): C andidit albicans^ cryptocooci and
/VjyrtMponi/i] ovale.
O fte n the skin o f the face, neck. hands and
b u tto c k s ca rrie s p a th o g e n ic h e m o ly tic
streptococci and staphylococci. Penicillin resistant
staphylococci are seen in individuals w orking in
hospitals-
l i i i i r frequently harbours SrapJi atirecj.s anJ
forms a reservoir Ibr cross infection.
T h e conjunctiva is relatively free from organisms
due to the flush ing action o f tears. I T lc piedom i nant
o r g a n is m s o f th e eye are d ip h th e r o id s
(C ufyiiehm 'tetium xerosis), M o ra x e lla species,
staphylococci and nonhemolytic streptococci,
T h e floor o f the nose harbours co ryaeb aeterii,
S tap h y lo co cci and stre p to co cci. H a r m o jJ liJ u v
species and A ft iruvcM. i /:eccjrtitCit may also be seen.
T h e A w p h a iy T u oftfac infant is Sterile at birth
b u t, w ith in 2 - 3 d a p after b irth , acquires the
com m on commensal flora and the pathogenic flora
carried hv the m other and the at tend ants. T h e
nasopharynx t an he co n sid e rs! the natural habitat
o f the com m on pathogenic bacteria w hich cause
infect iuns o f the nose, throat, bronchi and lungs-
C e r t a in G r a m n e g ativ e o rg a n is m s fro m the
intestinal tract such as Pseudornrijoas iirnrgurosj,
E .foli, paracolons and Pm teus are also occasionally
Found in normal persons. After penicillin therapy
they may be the predom inant flora.
T h e m o uth contains a plethora o f organism s —
pigmented and nnnjugm ented micrococci, some o f
w hich are aerobic, G ra m positive aerobic spore
bearing bacilli, colitorm s, Proteus and lacrobacilli,
T h e gum pockets between the teeth, and the crypts
o f the tonsils have a wide spectrum o f anaerobic
flo ra* an ae ro b ic m icrococci, m icroaerophilic and
anaerobic streptococci, vibrios, fusiform b acilli,
corynebactcfium species, Actinomyces, Leptuthrix,
mycoplasma, neisseria, and haaeroidcs arc all found
in varying extents. A m o n g Inngi, C a n d id a and
geotrichum have hcen reported.
T h e mouth o f the Infant is not sterile ^t birth.
It generally contains the same types o f organisms
in about rhe same relative numbers as those present
in tlie m o th e r’s v a g in a , th at is a m ix tu re o f
m ic ro c o c c i, stre p to co cci, col I Form b a c illi an d
D o d e rlie n s bacilli. T h e se organism s d im inish in
number during the first 2—5 days after birth and
are replaced by the types o f bacteria present in the
mouth o f the mother and nurse.
W ith in \2 hours after birth alpha hem olytic
streptococci ate found in the upper respiratniy tract
a n d becom e the d o m in a n t o rg a n is m s o f the
oropharynx and remain so for life. In the pharynx
and Trachea, flora sim ilar to that o f the m outh
establish themselves, Few bacteria are found in
normal bronchi. Sm aller bronchi and alveoli are
norm ally sterile,
In 8 0 -9 0 percent newborn infants, the m econium
is sterile but in 1 0 -2 0 per cent a tew organism s,
probahlv acquired [luring labour, may be present.
In al Leases, w ithin 4 -2 4 hours of birth an intestinal
flora is established partly from below and partly by
invasion from above. In breast fed children ike
intestine contains lactobarilli ( L . bifriAis constituting
99 per cent o f total o rg an ism s in the feces),
C o p y rig h te d m aterial
 4 Manna) Microbial Flora d dra Human Body »
entero co cci, colon bacilli and staphylococci. In
artifoinlly fed (bottle fed) children L . aiiitopAi/us1
and uolon bacilli and in p an by enterococci, G ra m
positive aerobic and anaerobic bacilli. W it h the
change o f food to the adult pattern, the flora change.
D ie t has a m a rked in flu e n ce on the relative
com position o f the intestinal and fecal flora.
I n the normal adult, the microorganisms on the
surface o f the esophageal w all are those swallowed
w ith s . l I l v .i and food. Because o f the |nw pj l ot
the stomachy it is virtually sterile except soon after
eating. In patients w ith carcin o m a o f the
stom ach o r achlorhydria Dr pyloric obstruction,
there is proliferation o f G ra m positive cocci and
b acilli.
T h e number o f bacteria increases progressively
b e y o n d the d u o d en u m , to the an Io n , b e in g
co m p act ively low i n. the small i u te ri ne. In the adult
duodenum there are l t f - 1 0 6 bacteria per gram , in
the jejunum and proximal ileum lQ ^ -lf l1 bacteria
pet gram , and i=i the lower ileum and cccum
l O ' - l O 14 bacteria per gram o f contents. In the
d u o d e n u m and up p er ile u m , la c to b a c illi and
enterococci predom inate but in the lower ileum
and cecum the flora resemble the h e a l flora. There
are about 10 " bacteria per gnam o f contents in the
colon and rectum, consti rut i nig 1 0 -2 0 per cent o f
the fecal mass In the adult no rm al colon, the
resident bacterial flora are mostly {9 6 -9 9 per cent)
anaerohes — a n e ro b lc streptococci., an ae ro b ic
lactobacilli, d o stii.lin , and bactenaidcs and about
1 - 4 per cent aerobes— enterococci, coliform s, and
s m a ll n u m b e rs o f P r o te u s , P seu d o m o n as,
l a c tubai. illi, m y c o p l a s m a , C a n d i d a a n d nrl u- ts.
N o r m \u F lor a o f i h r
G en it o u r in a r y T r a c t
A fy cn h a E tc riiin i am cjptiatiE^ a harmless cou::iLL":isal,
is found io the sm egm a o f the genitalia o f bath
m en and women. T h is may, by its presence i:n the
voided specimens o f urine, cause confusion. From
apparently no rm al m en, aerobic and anaerobic
bacteria can be cultured from a high proportion,
601
in c lu d in g la c to b a c illi. G i r d . v&ginali&t n lp h i
hem olytic streptococci and B a cttra d e s species.
Chlaiti. tr&chum&trt and Ureipltatna rjreaJyrjcrini
may also be present. T h e female urethra is cither
sterile or contains a few G ra m positive cocci.
T h e vulva o f the newborn child is sterile but
afte r 24 h o u rs it a c q u ire s a v a rie d flo ra o f
nonpathogenic organisms from the sMn, vagina and
intestines. T h e nature n f the flora in the vagina
depends on the p H o f its secretions and its enzyme
co ntent. I n the first 2 4 h o u rs it is invaded by
m icrococci, enterococci and diphtheroids. In 2 -3
d a y s , th e m a te rn a l e s trin in d u ce s g ly co g e n
depuSi rinn i n the vaginal ep i the! i u m .' Lb is faci Ii t n c ;
the growth o f a lactobacillus (Coderlien's bacillus)
w hich produces acid from glycogen, and the flora
for a few weeks is sim ilar to that o f the adult. After
the passively transferred cstrin has been eliminated
in the urine, the glycogen disappears, along with
D o d e rlic n 't b a cillu s and the p H o f the vagina
becomes alkaline. T h is brings about a change in
the flora to m icrococci, alpha and nonhem olytic
streptococci, coliforms and diphtheroids. A t puberty
the glycogen reappears and the p H changes to acid
due to the metabolic activity o f Doderhcn's bacilli,
j T” . l i and yeasts. T h is change probably helps 111
the prevention o f colonisation by possible harmful
m icro organism s. D u r in g pregnancy there is an
increase In Staphylococcus epidermidis, Doderlien s
bacilli and yeasts. O ccasionally other members o f
the intestinal flora may be present. A fter menopause,
the flora resembles that found before puberty. T h e
normal vaginal flora often includes anaerobic cocci
and hacllli, Liter ia, anaerobic streptococci, mimeae,
mycoplasma, Gardnerclla vaginalis, neisseriae and
spirochetes.
It v C T K R IA IN T H H B l . i ' " D MMLj
T issues
T h e com m ensals from the no rm al flora o f the
m outh, nasopharynx and intestinal tract may get
into the blood and tissues- T h e y are usually quickly
elim inated by the normal defence mechanisms o f
C o p y rig h te d m aterial
Hidden page
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Durham's tubes contain F . coti- From the number
o f positive tubes obtained, results are read off the
probability ta 'es. Further confirm ation of the
presence o f £ . coli can be obtained by testing for
indole production and citrate utilisation.
A m easured
volume oi water is filtered through a m illipnrc filter.
A ll the hacteria present are rctaLncd opi its surface.
Il ls placed on suitable m edia face upwards, aud
incubated at the appropriate temperature, and the
Colonies that develop cm rhe surface o f the membrane
arc co u n te d . A fte r 1 3 hours o f in cu b atio n the
presumptive coliform counts and E , coli counts can
be directly made.
Subcultures are made from all the positive bottles
in the p re s u m p tiv e c o lif o r m test in to tubes
c o n ta in in g 5 m l o f glu co se azide b ro th . T h e
presence o f Str, &cvsL's is indicated by the production
o f acid in the medium w ithin I S hours at 45 ~C.
The p o s itiv e tubes s h o u ld he p la te d o n to
M acConkev's agar for ronfirm atior.
M ilitp o re m em brane technique can also be
adopted for this purpose.
This
is tested by incubating varying quantities o f the
water in litm us m ilk m edium (anaerobically) at
?>1 ('C fo r five d ays an d lo o k in g for sto rm y
fermentation.
U nder
special circumstances, specific pathogens such as
typhoid bacilli or cholera vibrios may have to be
looked for in water. T h is used to be done by adding
the water samples to tenfold concentrated liquid
m e d ia , in c u b a t in g and sub c u lt u r in g o nto
appro priate so lid m edia. A sim p ler an d m ore
sensitive m ethod is ro filte r fbc w ater sam ple
through membrane Filters and incubarc the filters
on appropriate solid media.
M e t h o d s are a v a ila b le fo r the iso 1 1 1 io n o f
enteroviruses and other cytopathogeme viruses from
warer bur thev do nof form part o f routine testing.
A s a general rule, it is assumed that the viruses in
water arc destroyed by ch lo rin a tio n , w hen the
concentration o f free residual chlorine is at least
0.5 m g per litre, for a m inim um contact period o f
30 minutes at p H below K and a turbidity o f
t nephalom ctric turbidity unit or less.
E n ta m o e b a histolytica, f in ir d ia sp e c ie s und
Sa/antrdj'um coir can contaminate drinking water
However, there is no good indicator for protozoal
'Contam ination o f water. C o lifo rm counts arc not
reliable as indicators o f protozoal contam ination
of chlorinated water as they are more resistant to
chlorine than are coliforms-
These can be classified as below:
T h e com m onest
are lactic streptococci including Sir. factis and 5 tr
faecaJIs. I jcto b aciU i are also found. I'h cse ferment
lactone in the m ilk, producing acids, mainly lactic
acids, w hich lead to the form ation o f a sm ooth
gelatinous curd.
T h e iC consist
o f alkaligercs spp, some aerobic spore beaten; and
Achromobacter. These render the m ilk alkaline.
C o lif o r m b a c illi
are the commonest. O thers are Cl. perfringens and
CL buJvncujTt. A cid and gas. are produced. A smooth
gelatinous curd riddled with gas bubbles ls formed,
C o lifo rm bacilli ire responsible for the ropincss in
m ilk .
S p o r e -b e a r in g
aerobes such as B . subfiles and B. cereus, Proteus
vu^arfe, staphylococci and micrococci come under
this category.
Bacteria w h ich produce no
v isib le change in m ilk are calle d inert. T h e se
C o p y rig h te d m aterial
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 * B*c-.eriQlQ?y d Water, Mmk find Air ► 609

E:LiriliLT Reading

Lmtcn A l l and H M Dick (eda). 1990. Bactcm in the Environment. In Topley &. Wifsons iVirttJpJts : Bacteriology,
Virology and fmmujirr^ Vol. I General BjcttrioLo^j' and Immunity. Been inn B. 2 11-2 9 1. London: Edward Arnold.
World I f talrh Organization, Genevan G'urdWj'n« tb r D rinicing Winer Q uiliiy.
Vo], I. Recommendations (1983}
VftL l l HeaJih Criteria ( 1 W )
Vol 1[[ Drinking water qua]'ry control in small rommun:tysupplirs (1995).

C o p y rig h te d m aterial
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 1- k ■\ ' t / p

" . r ; : : ........... ; ; The routine method

ofJiagnos-LH lh bv the exam Lm::; kO H .
S crap io g l are taken from th r ringworm
lesions. T h e speum cn is mLv 1 ivlrh a drop f l U %
K O H on a slide, and after placing .l envershp,
preparation is gently heated tu mi
h l It-mscopy reveal - bra nched pt .lu i \ pli iu.-
6 5 .5) . Selection o f infected 1 . s.Lmiji.i on pi
facilitated bv exposure to I A lLc.rH W o o d s lamp).
Infected hair will be tluorencvi : :■ ■ ■ ■ i- :
infectio n may be d istinguished in a c t m ounts,
VctirthrL** in w hich iuthresp* res arc seen as a
sheath surro unding the h ah and 'endorl ■ \ in
w hich the spores arc ins id* iht hau sh ft ■ F i^.
6 5 .6 ) . D em on situation o f the lim g u sin lu ll may ht
d ifficu lt and may be possible i lv nib I n =i:
with K O H for a day or tw it
Species identification is po.^jbk ,Jc by odititc
cjrannin a tio n . S p e c im e n s are in o c u la te d o n
S a b o u r n u d ’s m e d iu m (w ith a n t ib io t ic s an d
CVC[-0 hexiitlide) acid incubated at n)otti ten]in.-rature.

Fly 65,-1 TrldigphjiL-i ruftfrunu culture mom:.: shawl

mi e rr.; on Id: 3 along • k r t t i hjyphftt, ; -r t la
^iotfrilc ■ «:tS11g o t o r.11! '%

C o p y rig h te d m aterial
 Topical antitungal agents are usual lv
effective. 7? rubram infections may be resistant to
treatment. Oral griseofulvin is the drug o f choice.

Tinea capitis M jcraqnnifn any species,

Trichophyton most species
C a n d id a s]! ^candidiasis m oniliasis) is an infection
FaYUS T. schiKnleinii, T. YTaEmum,
M . gypsoim of the skin, m ucosa, and rarely o f the internal
T inea barbae T. Tubrum, T.menrajpnphvtes, organs, caused by a yeaHtlike— fungus C a n d id a
T. verrucostrin albicans, and occasionally by o tter C a n d id a species,
Titita irnbnVdra T curroer?rrj\Tim C andida albicans is an o void or sp h e rica l
Tinea njfjMriu T. fubrunt aitd any o itw budding cell, w hich produces pseudomycelia both
dermatophyte
1'.cruris
in culture and in tihsueR {F ig . 65,7), C a n d id a species
ELflcccveom, T. nrfmim
T. fWifo 71 ruftfurn, /.'. dbettisturi arc normal inhabitants o f the skin and mucosa.
Ectothrix hair A fkit^H K am species. Candidosis is an Opjvrtunistic endogenous infection,
infection T. fXibfum, Tmerrf^Tqpfjiter the com m onest predisposing factor being diabetes.
Kndnrhrix hair T. schoenleinii, T. toruttran^ Cutaneous candidosis may be inDertriginous. or
infection T ndkcArA
fianmvch iat. T h e f<>rm cr is a n c rythe mitOUfl, sealing
or moist lesion w ith sharply demarcated borders,
Grow th is slow .and colonics may appear only in where papular lesions are most prom inent,T h e sites
1 - 3 wcclcs. affected lire those where the skin is macerated by
D e r m a t o p h y t o s is o ccu rs p ersp i ra t i o n— g f o i n , pier in e u m , a* i 11 a e and
throughout The world but certain types o f disease infra mammary folds. Paronychia and onychia are
imd some species ol Jungi show geographies ]|y seen in occupations th.ir lead to frequent immersion
restricted distribution. Social and cultural patterns o f the hands in, water.
also influence derm atuphytoses. T in e a pedis, so Com m on m u co sa l le sio n s are vaginitis
common in the temperate climates where all wear characterised by an a cid ic discharge and found
shoes ]t rare m die Lrop]CH where most walk barefoot. frequently in pregnancy, and and thrush found
M an y factors, such m age, hormoncR and intercunent com m only in bottle fed infants and the aged and
discascH, affect the susceptibility to dermatophytosis. debilitated. C n w n y white patches appear on the
D e p e n d in g on th e ir n a tu ra l h a b it a t, tongue or huecnl mucosa, that leave ji red OOtfifig
dermatophyres m ay be classfied as authrupophilic, surface on removal.
so ophilic anil grophlliL: species. H um an beings arc Intestinal candidosis is a frequent sequel to oral
rbc m a in o r o n ly h o sts fo r a n t h io p o p h ilic antibiotic ihcrapv and may present as diarrhea not
dermatophytes. T rubfum h E . floccosnm and jVf. re sp o n d in g to treatm e n t. B ro n c h o p u lm o n a ry
dudouiitsi are exam ple 5 . T h e y cause m ild hut candidosiB if seen j & a rare com plication of pre­
c h ro n ic lesio n s- Z o o p b ilic species are natural existing pulmonary or systemic disease. System i,
partsiten o f anim als. Exam ples arc v m in m jm infections, such as septicem ia, endocarditis and
in cattle and A /, cams: in Jogs and cats. H u m a n m eningitis may occur js terminal com plications in
in fe ctio n s w ith zjQcjphiiic derm atophytes cause severe generalised diseases such as leukem ia and
severe inflam m ation but are more readily curable. in persons on p ro lo nged im m u n o su p p re ssio n .
G e o p h ilic species, which occur naturally in s o il, C a n iliila granulom a and chronic mucocutaneous
arc relatively less pathogenic lor hum an beings. C a n d id ia sis arc serious m a n ifestatio n s seen in
Examples are M . gyp&cwn and T. ajellau'. im m unodefi cie ncies.

C o p y rig h te d m aterial
 therefore, o f greater significance. Clo tures can be
obtained readily on Sahuu mud's and nn ordinary
bacteriological culture media. C o lo n ie s arc creamy
white, smooth and w ith a yeasty odour. C a n d id a
albicans can he id en tified from Older C a n d id a
sp ecie s [C . S « l£ a to Jcfoe, C . tropica lis, C.
p sc■!jdotropidliSf C . kmsci' C . guilliermondii, C r
p itn p s ilo s is , C . visw xnarhii) by g ro w th
chai^tcristicB and sugar .L--y-i milaCioi i mid lenrencatiurt
rests. C j/hhun.s alone forms chlamydospores on
com mejil Jtgar cultttnfs at 2 0 * C . A rapid method
o f identifying C . is based on its ability 10

form germ tubes w ithin two future when incubated

in hu m an serum at 3 7 C ( R e y n o ld s -B r a u d e
phenomenon),
A k u l i n i n s appear in the sera o f patients but as
thee art tmjucnl in normal persons also, they arE
not helpful in diagnosis. Delayed hypcrtcrtsiliviiy
to Candida is so universal that sfcin testing with
Candida extracts is used as an indicator nit the
functional integrity o f celt mediated im m unity.
M a n a g e m e n t o f c a n d id o s is it m a in ly by
rem oving the predisposing causes. A ll C a n d id a
strains arc sensitive to Ny&tatin but as it is poorly
absorbed from the gut, it is nor useful in systemic
diseases. A m p h o tericin B , S-fluomocyTOsini and
c lu tr in ]a z o le m ay be used for d is s e m in a te d
candidosis.

Deep mveotic infections may be classified as those

that a heel m ainly or exclusively the subcutaneous
tissues (subcutaneous or intermediate mycosis) and
those that involve the internal organs (dee}’ sealed
■ or systemic mycoses).

can be established by microscopy and ] . M ycotic mycetoma

vulture. W r t film s or G r a in stained smears from 2 . Chrom oblasrom ycosis
le&iorts or exudates show budding G u r u positive H. Sporotrichosis
Otlll- A h C a n d id a can be seen on normal skin or 4. Rhi]ios}KiridLosis
mucova as Well, only its abundant presence is o f 5, Subcutaneous phycomycoHS
s ig n ific a n c e D c m o U t r it io n o f m ycelial forms k~yJ '■ Yrli
indicates colonisation and tissue invasion and is, I . Cryptococcosis

C opyrighted m aterial
Hidden page
Hidden page

R h in o Spur id iu Sis ]S a c h ro n ic gran u lo m ato u s
disease characterised hv the developm ent of
friable polyps, usually confined to the nose, mouth
or fly? but rarely seen on the genitalia or other
mucous membranes. T h o u g h the disease was first
identified in Argentina, the large m ajority n f cases
come from In d ia and Sri Lanka. W h ile the disease
is g e n e rally eon fin ed to m u co u s m em branes*
hem atogenous dissem ination has been recorded
very rarely.
E b io lo g ic a lly the lesion it eompused o f large
numbers o f timga] spherules embedded in a stroma
n f connective tissue and capillaries. T h e spherules
are U) 2(H) ptn ill diameter and contain thousands
o f endosfjures (F ig 65.1(1).
T h e causative fungus Khrnos/W j'dhJni Secbcri
has n o t been cultivated in m edia. T h e mode of"
in fe c tio n is not kn o w n th o u g h in fe c tio n is
believed to O riginate fro m stagnant water or aquatic
life.
I n this condition, originally reported from Indonesia
and subsequently identified in m any A s ia n and
A frican countries* a painless subcutaneous nodule
develops w hich enlarges to involve a whole lim b
or large arras of the body. T h e causative agent is
flasidj'ofrn/ti.s hapfosporus, a saprophytic
pbycomyeetc found in decaying vegetation and in
the intestines o f many reptiles .ind amphibians. It
has hern suggested that the in feed on may be
acquired by insect bites.
Cryptococcosis (torulosis) is a subacute or vh runic
in fe c tio n cau se d b y the yeast C r y p f o c o c c u s
ncofoimans. It is a round or *>vi iid budding cell, 4 -
20 pm in diameter, w ith apron line iu polysaccharide
capsule (Fig- 6 5 .1 1 ) . It is ,l soil saprophyte [ind is
particularly ahundant in the f c « S o f pigeons and
o th er birds,
Infection is usually acquired by inhalation hu t
may sometimes be through the skin or mucosa. M ost
in fe c tio n s arc a s y m p to m a tic . P u lm o n a r y
cryptococcosis may Lead to a m ild pneum onitis. As
no calcification occurs, healed pulm onary lesions
are not evident radiologically. D isscin itiatio n o f
infection leads to visceral, cutaneous and meningeal
disease. Visceral forms simulate tuberculosis and
C o p y rig h te d m aterial

cancer cl Lucidly Bones and joints may he in w Iv n L
Cutaneous atyptocoCco&is varies from small ulcers
ro large granulomas, C n p iu c o c c u l m eningitis is the
m m t serious type o f infection and can resemble
tuberculous or other chronic types o f m eningitis.
Its onset It ini; id loll h and the course slow am!
progressive, U is often seen in A I D S .
D iag n o sis is established by dem onstration o f
capsulated, budding yeast cells in the lesions and
by culture. T h e capsules stand out in In d ia ink
p re p a ra tio n s - T h e fu n g u s g ro w s r e a d ily on
Sabouraud's agar form ing im o a d i, m ucokl, cream
coloured colonies. T h e ability to gmw at 3 7 ° C
and hydrolyse urea differentiates C - mrofojTDilfl.s from
noApathogcnic cryptw-occi, Pathogenicity can be
dem onstrated by intracerebral or intraperitoneal
in o c u la tio n in to m ic e , w h ic h d e ve lo p a fatal
infection. C apsu latcd htidding yeast cells can be
demonstrated in the brain o f infected mice.
Tw o perfect stages o f the fungus have been
discovered.They belong COthe elasi; Bimdiornycetes
and have been termed FlfabsudlcUa f]Coftmii2DS
and F. busilispora,
hour serological types o f eryp tn co cu l capnular
p o ly in c d ia rid e - A , B , C and D - have been
identified. Dem onstration o f the capsular antigen
by p recip itatio n can som etim es be valuable in
diagnosing some cases (if o y p tK O tc a l m eningitis,
when the C S E is negative by smear and culture.
Am photericin li, D-Eluorocytosinc. clotrimazole and
miconazole are usefol in the treatment o f the disease.
Cryptococcosis is worldwide in distribution. A s
it Was originally reported from Europe, it used to
he known as ‘Eu ro p e an blastom ycosis'. Several
cases of cryptococcosis have been identified in In dia,
this being the only deep mycosis comm on in this
C o p y rig h te d m aterial
Hidden page
Hidden page
 A m p h o t e r ic in B has been fo und useful in
therapy.
. - * \- . _ J -. i_
Som e saprophytic fungi w hich are ubiquitous in
the environment are important in medical mycology
for two nelsons, Firstly, they are common laboratory
co n ta m in a n ts on vulture m edia— ArpC/gpIlus,
/t n id J /in m , M o co r and Rhizopva species grow on
v irtu a lly anything, Secondly, they can produce
serfou* and even fatal infection in persons w ho are
o th e rw ise d t b ilit a t e d . A s p e r g illo s is
mucorutveotis ate important opportunistic systemic
mycoses.
i ,' ‘ T V ’ L f . 0 : '!'•
A spergill! and Ptniciltia constitute the comm onest
moulds seen on dam p bread or almost -ir.v other
organic nutter- O f the JOT odd species o f aspergill!,
A . fiimigztvs is highly pathogenic for turds, and
o ccasio n al!}' Causes invasive disease in hum an
h e in g s . A few o th e r sp ecie s m ay also cause
o p p o rtu n istic hu m an disease, T h e co m m onest
hum an disease caused by aspetgilli is otomycosis.
System ic aspergillosis occurs as the following
clinical types:
1 . Pulm onary aspergillosis
a. Aspergillus asthma
b- Bronchopulmonary aspergillosis
c. C o lo n isin g aspergillosis (Aspergilhrm a)
2 . Distfinliund aspergillosis
Aspergillus astlim a occurs in atopic individuals
following sensitisation to inhaled aSpergillus spores.
Er bro n ch o p u lm o n ary asp erg illo sis, the fungus
and grows w ithin the lumen ot the bronchioles, w hich
may he occluded by fungus plugs. T h e fungus can
be demonstrated in sputum. T h e condition is made
worse by the development o f hypersensitivity to the
fungus, C o lo n is in g aspergillosis usually develops
in p re-ejt]stin g p u lm o n ary ca v itie s, such as in
tuberculosis or cystic disease. The fungus grows
into large 'bails' (aspergilloma}. Surgical removal
becomes necessary as the disease com m only causes
massive hem optysis. In invasive aspergillosis, the
fungus actively invades the lung tissue. Disseminated
aspergillosis involving the brain, kidney and other
organs is a fatal com plication sometimes seen in
C o p y rig h te d m aterial
 - Medical Mycology * (0 5

debilitated patients on prolonged treatment w ith can be grow n e a sily on S a b u u r a u d - m e d iu m

antab]o'i-'sf sterui Js and aludunu: lLulio-.
D ia g n o s is m ay be m ade by m icro sco p ic
r'^LmionT'on and bjrculture. T l lc fungus p iw S rapidly
on culture media. Identification of Aspergillus is easy,
based on growth ebaracre nst i ch and morphology.
Aspcrgjhj have septate hyphae. Asexual conidia are
arranged in chains, carried on elongated cells called
‘stengmata, borne on the expanded ends (vt-nicies) o f
Coiiidiuphurts (Fig. 6 5.14 ).
A s aspergiUi are sueh comm on contam inants,
their dem onstration in exudates and bolaLion in
cultures have to be interpreted w ith care.
P K N K JIt-l.ItJS lM
t V n ic illiu m sp ecies h ave been v e ry
incrim inated in opportunistic hum an infections E
n u rn ctfe .'h as been reported to be an im portant
o p p o r t u n is t p a th o g e n in the H I V in fe c te d .
M em bers o f this genus can be identified by the
brashlikc arrangement o f ennidia (F ig . 6 5 .14 ).
M l cm tM y c o s is
M u co rm yco sis an invasive disease Caused by
lh
phvtOTTtveetes-, m ainly by species o f R hr/o pus,
M u co r and Absjdiia. Ir used to be a rare terminal
complication o f uncontrolled diabetes and other
chronic debilitating diseases. TTte incidence o f [he
disease has increased considerably as a result of
the widespread use o f an tibio tics, steroids and
anrLmetabolLics. T h e lung: are norm ally avim lenr
and are able to invade tissues only when general
resistance extremely low-
T h c prim ary infection is usually in the upper
respi ratory tract or nose, where the spores gcnt-inaie
and the m yceiia invade the adjacent tissues— the
orbit, sinuses and the brain. Prim ary :i Joction may
atsn occur in the lung, the fungL invading the arteries
to cause thrombosis and i nfart Lion. T h e disease is
fatal.
D iag n o sis is usually made during histological
examination o f autopsy material, by the presence
o f hrnad, nonseptate m yccLi m Issues. T h e fungi
w ithout cvclohcxim
JF ide. M u c o r shows branched
sporting iophorcs a risin g ran d o m ly along aerial
m y ce liu m . R h i/o id s are absent. R h iz o p u s has
rhizniids, an d sporangiophorc& arise in groups
directly above the rhizoids (Fig. 6 5.14 ).
( ) r<HH Y C O S IS
Otom ycosis is a fungal infection o f the external ear
It is a very com m on disease and is usually caused
by species o f aspeigilli (A . rwger, A . fumiga tus) and
p e n icillia. T h e sym ptom s are itch in g , p ain and
deafness. Secondary bacterid infection, com m only
d u e to P seu dom onas and P r o te u s , cau se s
s u p p u ra tio n . D ia g n o s is can be m ad e by
ra re ly dem onstration o f the fungi in scrapings and by
culture.
O c u l o m y c o s is
M y co tic keratitis usually follows corneal trauma.
Fungal spores colonise th*. injured tissue and initiate
an inflammatory reaction, leading to hypopyon ulcer
and endophthalm itis. I f not recognised and treated
earlv, en u cle atio n m av becom e necessary. T h e
widespread use o f corticoslcriods in ophthalm ology
has re su lte d in an in c re a s e d In c id e n c e o f
keratomycosis.
M a n y s a p ro p h y tic fu n g i c a n cause o cu la r
in fe c tio n , A sp er^ fJJu s sp ecies, Fusiriuni and
C a n d id a afbicaire being most often responsible.
D iag n o sis mav be made by exam ination of
deep scrapings. Superficial swabs may not show
the fungus. Lo ca l application o f amphotericin E ,
N y statin and F im a ric iu (N ata m ycin ) may be
useful.
M Y C O T IC T o i s d n i n g
M a n y fungi form poisonous substances. M ycotic
poisoning is o f two types - m yccrism in w hich a
fungus w hich is eaten for itself causes toxic effects
and m y no to x ic o s is ml w h ic h fu n g a l to x in s
contaminate Siimc article of toed.

C o p y rig h te d m aterial
£26 4 Texcboak of M icrohm jgy t

Rfl. 6 & M Common conMMtantt fungi!. culture m w n lt PvnJewrt™ showing the 'penteNluf' or Brush, consoling
of chains of i p o w U tm tfln g from tha «n d t ot short branches ol oonMIophores. A§pnglllUM showing
unbramched rr-s a p ^ te c linrtnuiJrg In a globose i:::leto twirling fhlplldo from which a r id
Chains o f c o n ld li. Mine or showing n d m ip t it t mycelium without rh iig id i {root I Ike s tru c tu n i]*
Sporangtoptiorei, ml Idi m ty be brtn eh at L:wiimte In targe g to c s i sjmrenria oorttaiisln; nutrarwre s^onc*
n .i?tya > showing noneaptvtt mycelium with rhliokK, lAnbmchfld spornnfle-iiwrft* erf is uppers' i rhiiald/.

Copyrighted material
 ^ M edical M ycology * 627

has been known from ancient times,
several variL-ti^s o f poisonous mushrooms having
been identified as inedible. M ycetism may cause
gastrointestinal disease, dermatitis or death. T h e
hallucinogenic agents (d-lysergic acid, psilocybin)
produced by the Psifacvfw species and other fungi
liave attracted much attention in recent years.
T h e best k n o w n m y rot ox in i 1- 'a fla to x in
produced by ^spergi.'firs flam s. It is frequently
present in mouldy foods. particularly in groundnuts,
co m and peas. It is highly conic to anim als and
birds, and probably to hum an beings as well. It can
cause hepatom as in d ucklings and rats, and its
possible carcinogenic effect i.n hum an beings has
caused great concern. T h e re have been several
reports ofaflatoxLCORis from In d ia, invok ing human
beings and anim als.
Ergotox]costs (ergotism ) is due to the toxic
alkaloids produced by the fungus C7avioeps purpurea,
w hile growing on the (fluting heads o f rye.

Fu rther I(c u llin g

Evans EC V et ,J (cds). ]989. Afedrcaf Mycology. Ojcford; Oxford Univicrcity Pfoxx-

Kilibltr CC et aJ. lSrtfi. fVihcipfer and Practice afCIinical Mycology* Chichester Wiley.
Kwuti-C Iimii; ; KJ utd JE Bcnnrrt V&2. Medical Mycology. Fhiladelpliur Lea and Fcbi^r-
Richardson MD and DW Wirnock 1993. Fun^a/ Infection. Oxford! Blackwell.
RLyp^n JW. I9&B. Medical Mi coiogy. Philadelphia! WB 5 (under;.

C o p y rig h te d m aterial
 Laboratory Control of Antimicrobial Therapy
A p art from fire exceptions like Strep, p r o g e n y
p a th o g e n ic b a cte ria e x h ib it very great stra in
v a ria tio n s in s u s c e p tib ility to a n tib io tic* and
ch e m o th e ra p e u tic agents. T h in is p a rtic u la rly
m arked in the case o f Staph, aureus and G ra m
n e g a tiv e b a c illi. T h e re fo re , lc is esse n tial to
determine die susceptibility ot" isolates oFparhogEniL
bacteria to antibiotics that are likely to be used in
treatment. A ntibiotic sensitivity tests are o f two types,
diffusion tents and dilution tests.
D iffu s io n test: H ere the drug is allowed to
diffuse through a solid medium so that a gradient
is established, the concent!ation being highest near
the site o f application o f the drug and decreasing
w ith distance. T h e test bacterium is Seeded Ort the
medium and its sensitivity ro the drag determined
from the inhibit ion o f its growth. Several methods
have been used for the application o f the drug. It
m ay be added to ditches or holes cut in the medium
or to hollow cylinders (H ead y cups) placed on it.
T h e method moat com m only employed is to use
filter paper discs, impregnated w ith antibiotics.
T h e djisc diffusion method uses filter paper discs,
fi.O rnm in diam eter, charged with appropriate
concentrations of the drags. T h e discs are stored
d ry in the co ld. T h e y m ay be prepared in the
laboratory or purchased com m ercially A suitable
dilution o f a broth culture or a broth suspension o f
the COST bacterium Js flooded on the surface o f a
solid m edium ( M u d lc r -H in t o n agar or nutrient
agar). T h e plate is tilted to ensure uniform spreading
and the excess broth pipetted o ff Inoculation may
a l » be performed by spreading w ith swabs. After
drying the plate (3 7 "C for 3 0 m ins), antibiotic
discs (four or five per 1 0 cm plate) arc applied w ith
Sterile forceps. A fte r overnight incubation, the
degree o f sensitivity is determ ined by m easuring
the zones o f inhibition o f growth around the discs.
Grow th wifi be inhibited around discs containing
antibiotics to w hich the bacterium is susceptible
but not around [hose to w hich it is resistant.
T h e d ia m e te r o f the zo ne o f in h ib it io n is
influenced by a variety of (actors, such as difftisibility
of the drug, the disc concentration, che nature and
composition o f the m edium , its thickness, presence
o f inhibitory nr stimulatory substances, p H m d rime
o f in c u b a t io n , [r is, th e re fo re , n ece ssa ry ro
standardise all the variables. It is also necessary to
check the potency o f the discs periodically using as
control a standard bacterium o f known sensitivity,
such as StapL aureus Oxford strain ( N C T C 6 5 7 1).
T h e re are several recommendations regarding
the antibiotic concentrations to be used in discs.
T h e K ir b y -B a u c r and the I C S m ethods are in
co m m o n use, T a b le 6 6 .1 sho w s the d is c
co n ce n tra tio n s and the c ritic a l zo ne sizes for
amiliiutins in comm on use.
A suitable method for routine use in diagnostic
laboratories is the technique originally described
by Stokes. T h is incorporates b u ilt -in co n tro ls
ag a in st m any variables and therefore provides
dependable results, A standard sensitive strain o f
bacterium is inoculated in the m iddle third o f the
culture plate. T h e standard Strains used are Staph.
aureus A T C C 2 5 9 7 3 . E< co b A T C C 2 5 9 2 2 or
Ps.aeruginoia A T C C 2 7 9 5 3 , depending on the
b acteriu m to he tested. T h e test bacteriu m is
C o p y rig h te d m aterial
Hidden page
630 * Msdtea! Mycology ►

in o cu lated . A fte r o v ern ig h t in cu b atio n , the
'minimum inhibitory concentration’ (M IC ) ii read
by noting the Inwest concentration o f the dray that
inhibits g ro w th . T h e "m inimum b actericid al
concentration (M B C ) is the lo w « t concentration
o f the drug th at kills the bacterium . I t can be
estimated by subculturing bom the broth tubes that
show no growth on to suitable solid media.
The 'agar dilution method is more convenient
when several strains are to be tested at die same
time. Here* serial dilutions o f the drug arc prepared
in agar and pound into plates. T h e advantage is
that many strains can be inoculated on each plate
contain jo g an antib iotic dilution. A u to m ated
versions o f sensitivity tests are available and are in
use in large laboratories.
A n t i b i o t i c m a s a y s in lundy f lu id * : T h ese
are required to verily w heth er adequate drug
wnccntrarinns are achieved in blood and other body
fluids, and to guard against excessive blood levels
of potentially toxic drugs. The assays are generally
done by making serial dilutions o f the specimen
and inoculating standard suspensions o f bacteria of
known M IC . Assays can also be done by the agar
diffusion m ethod- T h is depends on the direct
relationship between antibiotic concentration and
the diam eter o f the zone o f inhibition with a
Standard sensitive Strain (lt bacterium.

Further Rending
Locum V. 19Bb Antibiotics in laboratory M edicine. Baltimore: Williams and Wilkins.
Murray P et al (edi). 1993. M anual o f C iin in i hiicrobtohgy. Wuhinput: ASM PIh u
W illiams RJ and D] Hcymm n 199ft. Gontumncnt of antib lotic resistance. Science 2. '1. ! 151

C o p y rig h te d m aterial
 Immunoprophylaxis

An important contribution of microbiology to

medicine bun been immunisation, which is one of the
mo&t effective methods of controlling infectious
diseases. By systematic Active immunisation many
developed countries have virtually eliminated ‘vaccine A t birth1 - P- Pn P BCG, QPV-O
preventable diseases' {VT'D) such as diphtheria, 4 weeks ........... BCGJ,
pertussis, tetanus, measles, mumps, rubella, and l )P T -l,O P V -t
poliomyelitis. The global eradication of smallpox, of 10 weeks DPT-2r OFV-2
course,, has been the crowning glory of immunisation.
1-a Tvcct? □ P T-X OPV-3
Immunoprophylaxis may be in the form of
(I) routine immunisation, which forms part of basic '4 mnnriis ............ M e u ltt
health caroi or (2) immunisation of individuals nr mfl-nths r , n, P DPT, OPV
selected groups exposed to risk of specific infections. 5-A yetrt . ........... m ■
(r d m l entry.)
10 years ........... TT ■
Routine immunisation schedules have been
16 years ........... TTJ
developed for different countries, and modified from
For pr^plldl TT-1 4IT buottti
time to time, based on the prevalence of infectious
women '
diseases, their public health importance, availability
ut suitable vaccines, their cost benefit factors, and Clm mondi after T T - L T‘T -2
logistic?. In India, the branded Programme on
Immunisation (B P I) and the Universal Note: 1 Fuf iristiiiLtiuEij.1 births unity. OPV-0 is
Immunisation Programme (U1F) have been able addlcivnaJ, and ro t to be counted Tor
[be primary court* o f 1 doiej m ir in g ii
to afford protection for much of the target
6 w h Ici.
population against VPD$r 2. Only for infants raft ri>til BCG it
The National Immunisation Schedule in force birth.
1. A second dew o f DT to I k pvrn 1u
in India is shown in Table 67.1. children with, no d o n im *n tary evidence-
In India, EPI and UTP have led to a significant atf history o f p n n n n ' D P T Lm m uniutlcin.
+ A tcconcJ dcra of TT to he given after
decline in the recorded incidence ofVPDs, as well dM 'i ■■■i' 11'■ tt> (huK with no raurfti nr
as of infant and child mortality, for example, it has history of pfior DPT, D T TT
been reported that in 1992 alone, 1.7 million lives LiiuTnuiiiitijOJi.
i. to r prevention nf tetimu in the nconuti:
nf children under five years wctc saved, as compared p rim irilp but also in (lit m other
to the mortality figures in 1934, the year before
UIP was started.

C o p y rig h te d m aterial
f> 3 2 i Texlaook
Immunisation with three doses of OPV has rot
been consistently effective in India and other
developing countries, with hi^li rates of
seroconversion feilures. Thi- is sought in be met
through the strategy of lmop ujf rounds by giving
OPV to all the children in an area on the same day,
expect ing natural spread nfthe vao, ine vitus among
the children to reinforce immunisation. These
rounds arc preferably held during October to April,
as the polio season in India is from May to October,
wnh a peak in July-August,
I)iflcrenrcnuntrk3i‘r:iji1ov different iinmuiLifiatiOrt
schedules depending on their priorities.
1 n DI\ 10L Mr IMMLINJSATHJN
Vaccines offered under national programmes are
limited by economic considerations and so some
important vaccines may be omitted because they
are costly These maybe supplemented by individual
initiative, whenever possible.
H e p a titis H v a c c in e : Many developing
countries, including India, have hi.Lfh. cndcmicity for
this virus, Ffcnnaial transmission and acquisition of
the vims infection in the first five stars of Life are
common in such areas, in contrast to low endem ic
areas where infection is usually acquired in
adolescence or adulthood from sexual or household
contactst contaminated needles, blood or blood
products or occupational exposure. Besides the
morbidity and mortality due to acute and chion ic vims
infect ion „ chronic carriage which may be very
prolonged is itself a serious public health problem- It
has also become an economic problem as earners are
denied entry oremplcymcnt in many foreign countries.
Inclusion of the hepatitis B vaccine in routine
childhood immunisation will therefore be bemefitiaL
The feet that a quarter to half the adult dn&e of the
vaccine is adequate for children brings down the
cost, T ill it becomes part of the national
immunisation schedule, it would be desirable to
have the vaccine adm mistered To as m.mv children
and adults as possible by individual immurisarion
or through voluntary agent :cs, The recent reduction
& Microbiology ►
in cost of the vaccine as a result of indigenous
manufacture, has made mass vacanati on more feasible.
MM It V '.l l l i e l l1; The composite tneislet-
mumps- rubella vaccine is employed in the affluent
countries but in the developing countries only the
measles vaccine is given it nine months, the earliest
age when it is likely to be immunogenic in the
presence of maternal antibodies in the baby.
Whenevtr possible, a dose of M M R vaccine may
be beneficial at 16-24 months or later, not only to
reinforce immunity against measles but also to
protect against mumps and rubella.
V aricella v a ccin e : Chickcnpoz is very mild
disease in children, but in adults it can be serious
and even fatal. In most parts of the world, chickenpoK
Is very rare in adults, hut in some areas in the tropics
it is not uncommon. The age of incidence of
varicella is reported to be rising. Varicella vaccine
had been used Ear many years in
immunocompromised children. Recently, with the
development of a more stable and effective vaccine,
its scope has been extended for general use for
prevention of varicella and herpes zoster The live
attenuated vaccine is recommeded as a single
subcutaneous dose in children 9 months to 12 years
of age, and as 2 doses at an interval of at least 6 weeks,
in those older Pkegnancy is a contraindication.
Typhoid v a c c in e Typhoid fever continues to
be a major public health problem in the developing
countries. Immunisation agaiinst typhoid is a real
need, particularly in view of the spread of drug
resistant typhoid strains. The original typhoid
vaccine is not widely used because of its uncertain
benefit and frequent adverse reactions. Two recent
typhoid vaccines, the live oral G al-E mutant
vaccine and tire imectahlepurified Vi polysaccharide
vaccine may be acceptable because they offer
prolonged protection and are free from reactions.
They are recommended for immunisation of those
five years old or above and so may be employed at
school entry.
Immunoprophylaxis of individual diseases has
been described in the respective chapters.
C o p y rig h te d m aterial
Hidden page
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 T e ta n u s us a result of hospital infections is now
rare but should be kepr in mind and toxoid
administered to nonirnmumc patients before elective
surgery. Many cases of n e o n a t a l tetanus have
occurred due to the use of contaminated umbilical
cord ties.
Even with
adequate precautions, catheterisation in hospitals
leads to urinary infections in about two per cent;
with indwelling catheters, the rare gtn'S UptO fQ
per cent or more. E r «Jr, Pmfeus, Ps. jaeunyprora
and other Gram negative bacilli are the causative
agents. Mixed infection iicammofl. Infection can
he prevented by strict aseps is during catheterisation.
Indwelling catheters are to be used only when
unavuiliableh and then only with proper closed
drainage.
A s p ir a tio n in
unconscious patients and pulmonary ventilation nr
instrumen ration may lead to nosocomial pneumonia,
particularly in those with pre-existing
cardicijuilmonarv disease. Multidrug resistant Sziph,
aiJrrws and Gran negative bar.illi are the common
pathogens. Antibiotic treatment is unsatisfactory'.
Postural drainage ls useful in the prevention and
management of such (w s -
These may
be consequences of infectious at any sire but arc
camTotmlvcaused bv infected intravenous cannula?.
The longer the cannulae are Icept in situ, the greater
the risk o f infection. Gur-downs’ on the leg veins
in liirants or children with diarrhea generally get
left in place for long periods, the site being bathed
in diarrheal stools. Phlebitis sets in w ilt Consequent
bacteremia, Many a child admitted with diarrhea
thus dies of septicemia. Gram negative bacilli arc
the common pathogens, 'Cur-downs’ arc safer on
the arms than on legs. Intravenous rehydrarion in
diarrhea should be restricted to emergencies and
should be replaced bv oral fluids as early as possible.
Infection can he prevented by proper skin toiler
before 'cut-down' and rhe qsc of stain less stee!
needles instead of plastic canrmlac.
Stmph. cpidcrmidis bacteremia is seen commonly
in paticnls with artificial heart valves. Bacteremia in
those with valvular defects may lead to endocarditis.
Hospital infection may occur sporadically or as
outbreaks. Etiological diagnosis is by the routine
bacteriological methods of smear, culture,
identification and sensitivity testing. When an
outbreak occurs, the source should he identified
and eliminated. This requires the sampling of
possible sources of infection such as hospital
personnel, inanimate ohiecls, water, air nr food.
Typing of isolate - phage, hacteriocin, artibingrann
or biotyping - from cases and sites may indicate a
causal connection. Obvious examples of sources of
hospital outbreaks are nasal Carriage of
staphylococci by surgeons or pseudomonas growing
in hum! lotions. Carriers should he suitably treated.
Sterilisation techniques have to be tested. The
cause of infection may be a defective autoclave or
imprJiper techniques such as boiling infusion sets
in ward sterilisers. A careful analysis of the pattern
of Infection may often reveal the source hut
sometimes it dudes the most diligent search.
It must be emphasised that control of hospital
infection should be not merely a spasmodic exercise
10 be employed when an outbreak occurs but rather
a permanent ongoing activity in any large hospital,
Evoy major hospital should have 'infection control
teams1 consisting of microbiologist^, medical and
nursing staff and hospital administrators. Besides
investigating and controlling outbreaks, their
functions include formulating appropriate
guidelines tor admission, nursing and treatment of
infectious patimb, surveillance On sterilisation and
disinfectant practices, determining antibiotic
policies and immunisation schedules, and educating
patients and hospital personnel on infection control.
Such measures help in reducing the incidence of
hospital infections, even if they do not eliminate
them altogether.
C o p y rig h te d m aterial
 Unfortunately, in many hospitals, infection
control is attempted by resorting to more and more
ot antibiotics. This is not only futile bait may even
be positively harmful by encouraging selective
colonisation by multi resistant pathogens. In the tinal
analysis, prevention of hospital infection rests on a
proper undersLanding of aseptic practices and
meticulous attention to hygienic principles. Sir
William Osier's aphorism that 'soap„ water and
comntonsense are the best dis infectants" applies even
today in the context of hospital in feet ion.
Biomedical or hospital waste mean* any waste
generated during health care, research, resting or
related procedures on human beings or animals
conducted in hospital*, clinics, laboratories at
similar establishments. This is far more dangerous
and offensive than domestic waste, It contains
infectious or other hazardous materials that mav
injure, infect or otherwise harm patients, their
visitors., hospital personnel and the public at large
in several ways. Biomedical waste if kept untreated
would ferment, attract flies and other insects, birds
and animals, making the place filthy and unhygienic.
It contains ’sharps" such as needles at broken glass
that can cause injury and infection. Discarded waste
attracts ragpickers who may repack disposables or
drugs and sell diem. The waste may contain harmful
chemicals and radioactive materials. liquid wastes
can spread, seep into soil and contaminate wells
and tanlcs pscliuti ng them. Unless audully m anaged,
biomedical waste can be serious pollutants of soil,
water and air.
W ith public opinion rising against
environmental pollution, governments across the
world are forced In bring legal restraints, in this
area. The Govcrmert of India has promulgated the
Medical Waste {Management and Handling)
Rules, 1998 under which the persons who are in
charge of medical and other institutions where such
wastes ait generated (called ’occupiers'1) are held
legally responsible for maintaining the conditions
prescribed in the rules, which have Come into effect
from 1 January 200.'!. The occupiers have to obtain
due authorisation from the prescribed authority
after sellin g up the required waste management
facilities.
"lltc amount of waste generated in hospitals under
Indian conditions has been estimated as 1 to 2 kg
per bed per day. This is composed of different types
of infectious waste, not all of which is infections.
On an average about K5 per cent is harmless and
IS per rent hazardous.
Mutinies* waffle i> paper, cardboard, cartons,
flowers and Ordinary office, or kitchen waste akin
to domestic waste.
infections waste is anv waste likely to -carry anti
transmit any type of pathogenic microbes, Ifejs
includes human or animal tissue* or organs
removed at biopsy, surgery or autopsy, placenta anti
Ot Iter peuduCTfi of concepti i>n, any pathologi ca| fluid
or diachargcs, dressing, swabs and Other soiled
items, laboratory samples sent for microbiology,
pathology and biochemical tests, all microbial
cultures, used syringe needles, used scalped blades
and other sharps.
Nonfofeetfeivf hazudous waste may be chemical
(toxic,corrosive, inflammable, reactive and
otherwise injurious), radioactive (handling and
management of which arc under the direction of
the Bhibha Atomic Research C entre), and
pfum ura^gied (surplus nr time expired drugs) r
A primary prerequisite
for effective waste management is a clean and tidy
environment. Waste tends to accumulate in dirty
surrounding. The hospital and its premises should
be kept in a clean and hygienic condition. This
requires frequent soap and water washing, mopping
and good housekeeping.
The objectives o f hiowaste management are to
prevent harm resulting from waste, minimise its
volume, retrive reliable materials, and ensure safe
C o p y rig h te d m aterial
Hidden page
X
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643 4 TEXTBOOK OF MICHOGIOLOGV s
bil-.iili..:i.Liii . nii :ir: i:: .ir:j -.i[ii■11. pmHYDOn nl earns
I!■U]I I IL11-L!■L"iIi L.I i;KML|l . inheritance melitensis 549
Bartonella badllifamt i'- bkt>d tran-f.t-ion nctrtomae i
I:.lM. il-ITi I blue pus i mis i
bartonelkras (Carrions disease1 blue tongue virus 522. suis
hif kl h:"r--11I - hapttrSpOTJF I body louse . brucellins I
baridiomycetes 611, bobin antigen brucellosi- l
bwidioepores 611 Bollinger bodies buffalo |■1 1 469
kuiey bacillus 166 Bolivian hemorrhage itiw 569 bukholdcria
Ji Ltlli (lymphocytes) bone marrow cuttuit ccpacea
booster dow mallei
character''tin ol BordctHxngnj medium . 342 pseudomallti
growth factor (bgf) bnftietella Bonyanro^ra virus
mirunnan bronchisep: ica bunyaviridae 447r 522-24,
BCG vaccine . M>A, epiLlem::ilo^y 542 bunyavirus 447
bcdsonla 422 laboratory diagnose 142 Burkirr's lymphoma 574,
bejel 385 parnpertU'-i-
Bence Jones protein 1 pathiigenLCbly buret K7K 438
h im k.i milnilk ,i ’ pertussis (earlier Haemophilus huryeivibrius
bemaUooninirn c-Jilor^lc- perrusai -) Bu&ler^ selective media
hnji j glutmldehjde I Latin (FTl bystander cells
ImlaKmi i prophylaxis
beta propiolactone (BPL1 tmlmcnt c
536, 54ft, 564 variation caiclvit idae
BFT reaction (biological false Bornholm disease (epidemic camp reaction
positive) pleurodynia) r California encephalitis virus
hhanja virus 514 .177, LrCmnm.iNitMLTi'riurn grmulonXHh*
Bhatnagar strain burgdorferi 389 camelpos 46E, 471
bifidobacterium 1 durtnnii i campy BAF selector media
dtntiutft 1 epidemiology Campylobacter
bile soluhiliiy ten hermsii . dr^edi
biomedical waste disposal laboratory distgrucH1- 388 coli
bird flu- '■ morphology 390 conciscua i
bwnM(b sub-:i]ivylate 1 parked i fenneOiae
Bicntr virus ! padi"i:en:,':tL 391 fonu
BK virus 561 prophylaxis ! joj-.m;
black death rtcmnntu jydon '.H pylori)
bla5to|feiiic (n>i c) factcir (B P treatment 3B9 SpUtOfU'n I
MI-1 vin^roiti J03 canarypox 469
Candida i
blMtontyres dennaticidis 622 twtryomycosi- 402, 619 alkicans ,
blastomycosis botulism 61ft 612,
blastospoies 612 bovine spongiform encephalopathy 6L3v ' .627
guiUiermond: ■
blood groups 567
A and its subgroup tirtadvLirti 11 ktUiei r
parapSilcHtis
Abo hemolytic disease branhaciella 1
pseudotropicali*
Abo system branhamella catarrhaJis -410 steUaioidae
and diseases I hemy impicaJi--
cross matching t 1 .l L11:l1 1 purpuric fever (BPF) visw......1iLL
Lewis system breast cancer 1 Candida granuloma
medical applications of Brill-Zinsser disease 415 l:l11>.LilL.:1?B9,
mn system bromovinyl deaxyuridine (BVDU) 459 capnocyrophaga 41(1
'O’ group btowrniTi movement capnpox virus 469
“OH1(Bombay) ' hruceth i capsid 444, 445
Rh system a b o rtu s . capvomers .
ari Iibodies antigenic tfiuctuii capsuLr pnJysacchjridt
antigens bactcriophs^s , 33< :

C o p y rig h te d m aterial
 s
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 K

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se°ceH
 • INDEX >
drug retisranoe (R) facror Hitnmopcucvinnic 46i l: b--i|ilL.i--
mutaTion.il > enteric- fe w (typhoid fern) erysiplothrur rhusuipathiaL
tnmsk'i ±ble (e],q^i>ni:iL'iiih'.'hi>i^i bactci iophigr typing i erythema
dubos medium diagnosia Of carnets arthrlticum epidtmicuitl
dysgunniBgjobuljiKinia drug rL^i-UriiT (Hayerhill fever)
dysgonic epideminLogv ctuonicum migrahs (annular skin
khn:-j['.hTy dlugzrmix lesion) 369
prophylaxis muhifemK
E treatment tVyVrgrn-| 310, 31], 362
ilr]', ,it,[:i; l-iv- 4fl4 enterobftctei , 603, 635 LTb’ hriiciL ,347
early protei ns ic n g tw erythroblastosis fetalis
Eaton agent 393 cloacae erythivibtastovix virus I
ebertbella Cyphi enterobacreriaceae- (■nthrocyte (E) antibody (A) complex
Eh nuclear antigen 4S4 ■_■]lihmili aetrnj
ebola fever 569 enierococouf ayrhragenii: (dick, scarbbiul) ii ixin
echo viruses enterotcodn Escherichia l■>1;
eclipse phase 436, 465 CltBHMdnm 445d447,
Ecthyma |r;i]ijrre!nriKiim , 635 466, 602,635,
eawrhfii. hair inCniofi Clinical syndrome
Eczema herpeticum 476 enniudns i antigenic structure I
Edmonrton-Zagreb strain 520 enzooncs 526, 531 biochemical reactions
cdw arclbiclEa enzyme immunoassay (eia) clinical infeedmt
efferent inhibition enzyme linked irnmocujwrbcrtt astar cultural characteristics
egg yolk medium reactLon (ELL5A) 5Jt2r enCrroasjregative (EAEC) 1
Egyptian u]ctf enzyme multiplied immunoassay enrtrohemnrrhjgiL.- (EH EO 1
Ehrhch phenomenon technique (EMJ ]'!■ e-nteroLnvasLvr (E3EC) 1
Ehrlichia enzyme neutralisation tests SOS entKropiatlwgrnic (EPEC) I
SCnnctSU i-><:.no|bhhia, tropical 382 aiMoemgenic (ETEC) i
EichwaJd-S Llmaer efleci ■j■.:■nl11■.:■-]1111_ lIiciiili1jl-1il' luchin <bf enterotoiins, methods for
Ei'kman test 6d4 anaphylaxis (ECF-A) detection !
Eiknella coirodens > eosinophilic hyaline induskwi hi hJIls 473 virulence factors
devlTnimmvnodLfEjson (see epidemic (Khokhitt *
antigen-antibody reactions) diarrhea of infant mice (ED1M) C H fh o g irij, H S V
election mscrasoopv 43lr 56$, 572 ES$ (erythrocyte sensitising
570 diteases 1 substance) I
dek pcErlpirnhon hemorrhagic fever 525 esrhiomcnc
LIn iiLutaT^' txyJi: :- 422, 431, I Iwitooonmoohtidi (EKC) 4U6 ether 432, , , 563
elution 435, 503 nephritis (EN) I ^idivlnie .3-i r: inc tetrace-tic acid
EMJH modium 390 nephrosnuephritiF I (EDTA) I
EMRSA pimililis (tee also mumps) eubactenum
encephalic* 397, -p[curin.Lvma eugonic 'I
522, 523, 524,525, 535, polyarthritis 525 eukaryote , 1
539 typhus C\iltkli ['H]
L-tri1r j ] (junificU1 52 5 epiLlcrmiipliytnib 614 exanthem subnvm (roseola infantum
fatal 523 episodic angioneurotic edema 114 Hlinl] dittut) ;
tisv 477 episodic lymphopenia cdbliailvc toxin
blLvI[IC epitomes esophiala werneckii 612
encephalomyelitis epitope ' "! txotrtsin a
encephalopathy epizootics ,329,531,534, :
endemic diseases l Eptttiit-Bvr vim* , expanded programme on
e o d n iib L ' \ i r nzb-uLuj]) ty p h o ii-l 574, immunisation
L: Ld I--II li L lb |'lia r equine encephalitis virus, 523, 52 S, expanded n'heUa tyndrome 564
endothiix ba.iT infection equine rabies immune globulin experimcciral allergic
l=l: I■11 □ri-i (EH%) i encrphalomyelitis
Entamoeba histolynca , 635 ergotism (ergoioxicciiis) 627 Splint cultures 439
Entamoeba moritinaft.iliom 46] erwinia ortnapirimonary tuhcrculosih 360

C o p y rig h te d m aterial
 ndaiensis 321.331, 332, 146 versus host ieactkm 1S0
fab fragments 85 freeze diyinp 414 Gram stained smear technique 9,10
faenia tp 402 freeze etching 9 (Jravrs disease (see th^Totiwirosis)
fivus (TrichophvTon suhonleLnii) 610, Fid * test -426. J2S gntfjlh typing 2fl-4
614, 6l6 Freund's, adjuvant I4t>. 169, 171 grjsecdulvm 61b
k fi^jiidiil 85 Putin's method 205 Griets 1 1 irnte test 277
fcfci tubes 2 » fungi 143. 229.610-27 group-specific nucleoprotein antigens
fermentation 21 fi-Frtjn im p e r ld - li (d c u tfiffliL Y C e te V 577. 579
Fernandez's reaction 373 hyphomycrai) 6! 1 jrrawth inJiLbliion tesr 397, 390
fatlinuriTTTfJ AHC> n;Li'iii|i.ililiiEt', 1R9 fusiform bacilli 235, b00 Guamieri bodies 449, 470
Ffocw fowboctenuitl 267. 2b*. 2710 GLLiHai.n Kane lynditune fidi(ypathiL"
fibrinol^sins 5fl(T (UotpirutheBii) 388 p tJw ie iir itL i) 173.477
fibroma 46* n n H W (duunik geanulomata) 370,
173
fibronectin (frmdii^ protein) 127
Fime boufcmEieuse 414, 416 g*ffky 1 1
Filubasi J icUs b,ui1lispoT;i 671 qai-e mutant vaccine 642
Filobasidiella neofonmans 621 ganima^obulin 153, 15J haeitiEigogus spegamEiu: 529
fiLdes tear 334 jpittichnir 4*0 haemaph^salis
fikfenbe 448, 56? girijdm diwase (oftltHp] 522 leachi 416
riLtratinq 3? gitijrtm itiM 522. 5.44 spini^era 531
fimhfut 272r 273 gardnereUavagmaJi:, 229, 410-11, 6fll ticks 510-41
Ficf-J lugh Curtia syndrome 427 pas gangrene 248. 349. 251. 35 ? h^mupliilui 224, 333-38, finn
flageU;i (we bacterip, cell will) uHfflbii: myositis 252 acgypticus ^37
flavlvirus 447, 525-26 endogenous 254 a^ihrmphilui ,430
flm bxhiiuni 60-.1 propfryiwis and therapy 25b duereyi 337
■[LCFmigMM^hliCUIFL 4Ckt Ra^puk. syvtecEi -11. 269 haemolytiius 339
fleabome typhus. 4H GB virus 559 influenzae 333
flea ind« 22* Jennie tt^ineenne (see bacterid parahcmolyrieus -417
flesh catinp bftCKtia 2fJB generic)) parainfluenzae
Fletcher's medium 390 genital chLimydiasi.s427 panphroptuliis 336, 33S
flocculation ^4 genotjpLC muung 445 HaSkine's Vfcdnt 330
flurtuaTi(in test 55 gentamicin 403, 405 h iim a JB l
fluorescent antiliudy Test 104, 409 Rtji UTi-vhum 600- Ei:ilLh.-iUjcil:t:i IVAia^.ck (HP) bodie)
fn-pimenial antibody test 104. i^rman m ask ) (b{* alsu rubdk) 505 425,426, 426
Aijnrweitt dvr;- 351, .157 gcrmiidie 24 haJophiiic nbri(js 316
5-fluo.rouradj 390 ghoul focus 355 band, foot and mouth disease 497
S-fluorocytosint 617. 619, *21 giardu sp. 605 bantavinw 447, 5.14
flucr Etnin 4*9, 536, 54t C ilU H S Ftairi method 4 12. 417 bin run virus 543,
fcdticulius 589 gifirua s>um 424, 375, .TSb, .1KW, .190, haplotype 132
I'.w i it a (hof modeardrum) 618 396, 424, 426, +49. 47H, 4*3, 519, haptens SO. 81,83, 379
Fontana’s method 378, 540 llaid ohatiL il 379
food p o iw iif 352, 271, .116 Cbned a E V Kftui 330 ELdiLiiiLHi:.'b Eiiscase ilvEiiphjiltELoid
foot and mouth disease 606 glandular few S3, 170, 171, L89.3B2. goitre) 172
forbidden clones 149 +03. U l HasspH's corpuscles 11*
formaldehyde 30, 31. 243, 249, 2FK, globulin (see immunoglobulin) KAV vaLciTie 540
291,300,330,434. 502, 513, 513, gluon 182 biwar? vim? 544
548, 5.56 g^utmUetyfe 31. 2w, 283 EleatV niJtiplc :tuatuit techuxf^ .161
formol toxoid (fluid moeoid) Z38 glycnpfomein 185, 4.15, 504, Si I heat lahLle noun (HLT) 440
forscarnet (triaodnni GM (gamma markri >system 91 beat stable umin 274,2?9,26l, 314
pbusphunufointipte) 4*0 gmJtpoK 460 hfavy chain *6
forraiEiaiL anligui i 52.484 pnl^p apparatus 125 diaeasC 90
fowl plague 507 gui uxskljus (see d-.i i iiL'iancria) 1, 17, hela oe!l line +12,42$, 426,440, 444,
fowlpOK 46* 22,225 475.479, 5U, 516
F IlurjunJ prordn 5 16 gui iLnrlttiii 225 29 ! LI j cdb 2B7,425,42h, 475, +^, +96
fainlw iu (h:C JWfl) O piiiEL :r 5!b hdper vin»430
liiiicudli 331 graft 176-H0 hemadsorptun 4+0, 449.504,506,507
 i INDEX ► 647
hLim.yl.ihrtjri.-n J :J, hib PRP vaccine I Hunter: .in chancre 379
5K , hide porter’s disease ImlltHftnv licks 534
519. 5.1* I likiijima strain i hybridomas
inhiViiiiiai icj i .197, 19E, 504, hingf region , 37 hydrogen sulphide production test i
531, Hippolites paliippes 365 hydrophobia I
indirect . 196 Hiss' serum water medium h ill- iphobmpboni i ^lyFcaphnbu)
passive 129,397, histamine ronHSHpR jKftir(HbF) hypft tgK jyprimnvr
Lira! hiSfMjCytc (ice rTuCrtiphjge) hypenemii LvLry
Ik'NI l[t
jt 11
|..I ■ ; VI-- 11 ■- 1 : ■.nmpatlhilicy antigens ample
I- :I.iiiir ii- hiictH]plasma 621 classification of
hcmalj^iH capsulatum 623 delayed (or cell mediated)
hrmnly^s I dufmisii *23 immediate
.iI--1i:i , I'l'I m hisroplasmosis 623 immediate vs delayed 1
I I - Ivr-disease of newborn , alrican 623 types of mictions and their
histnry nf hactETMihTgy features
K'rTh.ii;i' fevrrs, viral 563 HIV inli ' lic'ii 39, hr.i■■>chy^uiiIiL-m (iin \edenia)
:1 : irrhi.ij |. fever v-'th renal H L A molecules 1 hjpoxanthme phwhorpbnyyl
H > nmt (HFRS) 563 alpha . chain- ■r..ri'_iT.i'i ( H F H l i
hendta virus i I I LA typing hypha 610
cell line 447 Hodgkin's disease , 599
hepadnavnus . 447 IpimiK vUVfrtiprtni r
hepa.ti.i'- i-mse-n 'horror midWtakuS1
]j. in!:s|cn
viral hepatite hospitil infection 614-39
!■:l-|i.i1 1 r:- type A (infectious hepatitis) common types of 635 iatrogenic infectu ns 6l4
diagnoti- and control of i 1CRC bnciilua i
(actors contribufrig to 634 Id i 1
hcpilri: A virus (HAV)
ErncTnhirdnfQ/ nf 634 idnsyuntlinr (IDU) I
hepatiiis type R (scriip bepati.is) 549
srcTiLivirtfp equipment .i::.l ■[Jhil-.l.-i M- (see dcrmatophyiids)
antigens and anritKxf e* 553
blbni1-. vie .lj. 52f-
hepatitis B virus 43Er techniques, testing of 614
and hepatocellular car;" noma hospital waste d'-posal i immune adherence
immune com plex (or I. >xnr complex
553,554 Hug£r-Llefsi:n'» EesI
hepatitis type C human body louse {pcdiculus diseases) ,
hepatitis type D (delta! 436, I bUBUdt corporis) 387, immune oytolysis
hum.lii diploid cell STnlio (l SIX'S' ■11 iiiminr * lirt.i Oj Iion
hepHtitis type E (E-NAN]!) 559
hepatitis type C virus 559 vaccine immune response
cellular : del wed hypersensitivity)
hepatitis, NANB 559 human embryonic kidney cells t
hepatiti- vaccines 549 human gammaglobulin 142,
effect ii! antibody
Iil'II-.-i'i'l!i;!.i: Cirt i—:-ni.; 553, human immunodddency virus
AIDS related virna I effect of malnutrition
554,572,
antigenic variation and diversity genes
hereditary angioneurotic edema 114.
erf 5R4 humoral
herpangiriB :ve-:cular phstryngh-l
i; m::;iim'■kvi'.'.i! abnormalities of in malignancy
herpes fehrilis 476
negative phase
herpes lr-:nru 539 Inirivirusta 584 primary
herpes ■ ’implex 476, 481 pathogEneiix 5S6 secondify
herpesviridae resistance I immune RNA
herpesviruses siies of iftolarion
immune system
herpesvirus simiae (B virus) structure of
ntunune tolerance
■-.■ii. 11 -■■i: I■:.. L1 1 1.■herpes virus I: human herpesvirus type
■Timur i-at on
herpes zoster (shingles, v-- uu ■ human leukocyte antip: n (HLA) 1
herpes zoster, ophthalmicus 481 complex cumhined 7E
herpnie whitlow 476 human papilloma virus (HPV) routine
hcnerophile antigen (see Faresman human table s immune globulin individual immuiii-n^on ■
antigen) schedules fer
(HRIjs)
helerotmphi human T cell leukemia vims 0 f T L V )
Lnamuruiy
acquired (adaptive ■
henons
herd 79
HFR cell 1 human T n-ll Jytnphotnq^: vli us-111 I
Lnnate

C o p y rig h te d m aterial
MS - TEXTBOOK O f MICROBIOLOGY ►
W 7SF upj.i rnii-ri bodies 1 morphology '
measurement of" 7¥ inrracyroplasniic onhomjnamrus and
immujKX^nghitinin (IK) intranuclear f pflramyMVMrUE
■immunodeficiency diseases 152—59 inclusion comiuncrivitis, neonatal form pandemic 5012, 5in
(Mbn^v 11 i-.iiitii-.iulc-ri ^i!:=■^i-.-s of 427 pathogenesis 50E:
tliiiifUiiriLWi of ]$3 indole production w$t prophylaxis 511
djiMtlcCS of complement 15 1. .i f Sporadic 509
disorders of phagotyrais infecrion treatment 511
secondary imrtmrKxiefidenciei classification of I types A .B,C5G 2h5&5f S ll
iinmunodiffiiEitm (prrci pi r.tfion in steady state Infection (ceDular interfrienoL 445
JS«I) ; injury) mtatnuna (UW) , 445,454, i
tadi j! i mnr unity interleukins (IIj
hnmLmoelectTnn rmcfoacopy modes of IransmiE ^on ■ ( intrauterine infections 69, 401
iinmunoelecttophoresifi microbial pathogenicity, iactora intravascular coagulation,
nitmumcmyinc t«r adhesion disseminated 114,
i:ftHiunoflvUie*C(n« !nt? h:i..i i-Tij.1 appendage; mdine
, , 3fi2, 3 9 0 , 3 9 2 , W7„ bacterial products araining
39S, ' .405, ", ", bacteriophages mdophones
.484, 514, 515„ .519, communicability ' n.ioifLuidinc (5-iodo-2^dcoxyuridinc)
513 infecting do$e 1 425, i
immunoiilobLns (IG) in vu H n ta isnanttgcm 1
In fill," ", ' ”, plasmids l TUAT (international union against
route of infection 1 tuberculosis) '
and BCnpy 1 to’cigeni vity ■ Iuat-LJ medium 356
antibody tiMte 99 hii Jiapc.J KtnrtieE of Ixodes dammim 369
serum IgA ^ (S lg A ) wdwtsun* 1 ixodid tids 419,531
lg D endotoxins ,i 1
Ig E , 153, . era toxins
i-uihiviiv. >s endotoxins
Ig P S V . t -'-i .111' sources of Japanese encephalidE 522, 52b
Japanese 'IV encephalitis 526
n r“ lj.rih,'.: 1JiTxhnrruT ifKttnnc I
infections, vinn
Ig M iW . . cl-iL iT.nLner jpv ■:( 459 Jc vdru^-
i- i
host responsc Jennet's coT*pcot vaccine
564, irnimimty in Jennet's net^erk hypotberis
iiHmLirti^nhulin gene sup^fiunily Lmmimoprophyiaxi' of
Jeiyl-Lynn strain 514
■f'[ i it . u |n1 il' i i i: lI i ilo g y Laboratory diagnosis
Jh virus 499
immunological paralysis nanieomunok^ical responses Job's syndrome
JrthrtL't hiiilhai fM'f M-
: mrri l-111 :■11 1ur-'-J-1 surveillance pathogenesis of i
pa. ™ tu b ? m J os i e)
immunofogica] tolerance screening of vims i
immunologically competenc ll-11 t^fcchnens to be sent todu^nosi-’
}: ny-:—\[i.|r_ h^vMn'hivily reaction
( IC C ) : “ r infectious muncuiudeofliE (gianduUr
iinraunolyni] test fera) S3. 3B2, K
imi nu niiriK Irio tests infective hepatitis (see hepatitis) K .mligens ,
i mu 11.1111iprftphyl jjti ■
■ inflammation KAF (jfe: oonglutinadon activating
LmmuncifluppressLvE agents illOlHAU factor)
immunotherapy in malignancy antigenic classification 505 killer: K■cells (see ADCC cdk)
imperial , 509 antigenic aEmetine 504 Kanagnwi phenomenon
1MVTC tests * antigenic variation 504 KapcvTs sarcoma , 592,
bidtunnon clinical features 506 5B9.
inclusion blcncmhca 427 epidemic 50S Kaposi's sincetljForm eruptions
inclusion bodies 425, 425 epidemiology 509 kwbn virw 534
acidophilic 1 hem aggju> ination KauifrnJuin-White scheme
basophilic 1 host rni^g? 506 KCN medium
Cowdry type A / B t immunity 508 Kekv Etta1ns 536
eosinophilic laboratory diagnose Kelley's medium 399

C o p y rig h te d m aterial

krjion 614
kingella 410
Kirby-Bauer method I laboratory diagnosis 391
kiHinp disease
klcbsielb
K l t t m - flirt bacillus
Ki.vh'i phenomenon I
Kochi postulates
Koch-wwlts bacillus
KipjiIik» spots 561
KtHThoFi medium 190
K^-er'f citrate medium
Koyhc'^ reagent/method
kuru , 367
kupffer cells
Kvasanur forest disease (KED)
522, 524
l
Lab-[^mLni kornmeiaa] meat
MWKt)
buev i D tP medium J42
hcfcfadlhjs
Lancefield groups
acid extraction method
Lan^erhirib cells
Large-Sachs group
bxu fever 569
bssa virus 447, 569
Lares fixation tear
httice hypothesis
bay leueocvne syndrome 15:1,
LD i i
LEbody
LE cell phenomenon 173,
leirionrlia
kl^iontHLTCi '■Ji'L-dW I
krtrLvLrtLjo 566
leporipcot virus 46B
lepra (foamy) cell i flJfTS)
lepra m aion
lepromin itit
Leproty . 367,
clarification 371
cultivation
tpidHHiali^ .172
immunity 17.2
bhruatcyry diagiwris 374
morphology i lumpy skin disease 469
prophclixi-
c-rikrj .n■.f 371
lcf:l:>'.]iir.i 3R9
antigenic properties 390
mineral dwtCKtiftict 19(1
epidemiology 392
« INDEX » &49
ictfmhrmnrrhagiu 399 jpnpfotytt tTansfonnatvm tests 372
interrogans 399 Jymptuxytosii producing factor 1
lymphocytic chotiomeningiiii (LCM)
serological diagnosis 392 virus 447,
morphology 390 lymphogranuloma
patliLigemril'v 391 inpiipale !
prophylaxis venereum (LGV) I
resistance 390 lymphoid organs, central
therapy bursa of fabricrus 1
leptospirosis 391-93 bursal lymphocyte*
leptouhtfbc i {B cells) 1
leptoirichia bursa dependent uvu
leucocjdin i thymus
WuJKyK aiti'.'.iting factor (1*A1,3 mesenchymal stem cells
leu&Tcytc difierendarion antigpii umi disease
leucocyte GfiPD deficiency thy anEigcius
leukosis sarcoma vims lymphoid organs, peripheral 1
iLnikiiCiiuuus bronchus associated Lymphoid
JevamLsole tissues {BALT)
LewnrhaL-Cole-Lillt (LCL) bodit v.lL-j .-.milij' c.lI Eviunlioiti tissues
Levinthafs Hgar medium 334, 33b, (GALT)
L forms exfbacteria lymph nodes 1
405, toilcos a-assoc inted Lymphoid
, .i ,i
jrT<t :ni,L'>pbsnia 1 tissues (MALT) 1
limes nul dose 1 mucosal (or H w taiy) tmmitEVt
li m a kv I I dose 1 system i
Jimulus polyphemus spleen . J
•
lipopolysaccturide {LPS) toxin germinal center, 1
ubjp^Mri ,xvt|iils, Ii^ or (ollii lf.L
[.n'si. huV£ inclusion bodies lynifitLokiiiL acrivaled killer (LAK)
l:i|Miinl cells . _J
Intern lymphomas 574
listeria monocymgenes EB associiiillI
llsucrioiis lymphomatosis
litmus milk test lymphopoicsu
liver infusion medb lymphortEicubr 5ystemhcells of 1
[.Hiefflcr'r methylene Wuc , 357 lymphocytes
T v . ;il:.rK, il '-L
Irftfffle/isemm slope
I lyrtlfli-'kiiTLs
long aciing thvroid stimulator
phagjosytic cells , 1
luu.]'injj ill 524, lymphcrtCain (TNK-beta)
louse of human body (pcdiculus lyophihsaition
htunanui eorpotii) ■ lysti^mL (.ynle 465
Lowelutein -Jeiuen (LJ) medium lysogpmc harturia
367 lytOgenic HitvtniM 465
Lode's tumour frqgs i lysogeny 465
LudlamV medium lysoocnisMiHi I
Ludwig's angina I lyxLtl 329
lysosomes
lupus vulgaris 362 JysrwiCaphin
lygrantim I lysoryme
Lyme disease 399 Kssaphobta (hydrophobiophobia) '•
lYrtiphadenOpathv usiHniiit<d virus BMPiiui 447, i
{LAV) 532 lytjc (or H'irulee: i I civic
lymphoblasts 'lysis fiom ivithout' 564
C o p y rig h te d m aterial
 t TEXTBOOK OF WOnOBOJOGY >
M 447, lethal dose [MLD- " '
MicConkrty's medium 563, reacting dose (MRD) 1
m.iL Li^■■-■:-|I-: -li.uv.i clinical features mink encephalopathy 567
miiihupo virus 569 laboratory diagnosis 5l9 mitogenic factor (MF)
::idL::ii;l'.:'liijli:LL:nij. ' vaccLoe 520 Mitsudj's ,.t .i:Il ;inr:|ti-t. 574
macnxonidia 612-14 virus Mirsudl's litt rrirlmr 574
macrophages . . . mediterranean fewer mixed leucocyte reariinn (MLR)
i i medusa head appearance (see bacillus)
Activated melioidosis 1 mixed lymphocyte culture (MLC l
ajt'MvflMWagKrei^njn factor meiiMjry uelh iT.nlrl'.ini'.i' .4 ]0
(MAF) mer.usgchoccemia molecular biology, basic principle of
in. blood (monocytes) mei inRococcu* (see N meningi tides.)
characterise ics of molecular ep:dem: ologj
cbemotacdc fortor (M CF) metabolic ■■■Ir.l■:■I■■-; 398, 1 IhuUlCutE}
iii'U/jiiHir'i iiL' ihiiiii^ factor IM1F) metachromadi Moduficum «mtagjosum
metachroma’ ic granules (see Babes- nmnm 1
mononuclear i Erna granules) moniliasis 389,
in tissue (hi-tiocytts.i methanamiiw silver stain 611 monkey fever 531
mad cow c£i ^eaae 567 TAttiUriotaKteriiL monkeypox 468, 471
madmrnmss (maduia foot) 40(2,619 meihyieise blue reducium ten monoclonal antibodks
madmcUa 618 606 mnnocyic (see macrophage)
maedi (progressive rmcumoni.il 567 merhyl red (MR) m-t monokines
MAI complex 366 MTadyean’s tth>.tion. monomorphism
major histocompatibility complex microaeraphilic bacteria i Mansur's CTTA medium
(MHC) microbial flora of human body, Morav-Axenfeld bacillus 410
antigen normal moraxella
gtrie* micrococci , r11 ■iLi !1i■.■:ri.■- 447,
j

II I' I-L'. lll'-

micnnonnLdia 6l2, tils. 6l4 iruoiganeLa
nealrKnor. micron majganii (formerly Proteus
major outer membrane protein microphafie mafgaii.ii! ■
(MOMP) mjcrasccpe mwpobolugiciJ index
maJasseiia furfur 612 dark field ■.link ground) , mould* (atgwtyillut, mucor) 607, 610,
malignancy 391,
i::uiiij!LL' response in direct examination mousepox , 46$
immunology of clectiun : M protei™, , , .
malignant edem i :iiierf=eme : 504. t
malignant pustule optical (or light) MS murker 494
MaUdn itit phase contrast , tnucofmjmsjH
nuhs ( m i pokin'-.liion mucot species 612,
mammary tumour virus (of mice!- miwMpwum 614 Mueller-Hinton med: tim .
munoLix rest Vi], rnicrotumours
Marburg disease 569 Middlebrook's medium ' mulriple myelcuTU
Muck's disease < m-.irr.in.,n inhibition factor (mifl mumps , 44k,
Mascm-Pfizer monkey mammary milk
Laoear ki'.l u: i; iloid'-al cumin jciiiil Wit, clinical feature1
rriail CfiTli diseases, niilkborne 60fi (■]iliIl-i■,i:
mastadenoviru- milker? undes (parawrinia) 466, immunity 514
Maxlcds method 1 472, H)6 laboratory diagnosis 514
rnayaro virus 522,524, 525 millionnajie nnokcule S9 properties of the virus
mazucclM w ane mima polymarpha 405 prophylaxis 514
Met ride's intratypic mimeae 405, i mumps orchids
marker 494 minimum bactericidal l■:iill~iiEt:iii>:sn imirine Imkosis virus 1
M l(.?mv cull Ilmc 425, (M BQ 1 mui ifl* typhus (ret endemic typhus)
Mnintwb-Eil Jea' jar minimum hemolytic dose(MHD) murray valley enuphalLtL* virus 526
Mdeod's medium min'.mum nJtKting doeefMID) mus musculus )
measles , iiitutMiory concetirradon :MK'i muCation
miiogenE 142

C o p y rig h te d m aterial
□

i*-.
E e 6
652 i TEXTBO O K O F MICROBIOLOGY *
papilloma virus 574 pettggnini medium plasmids
pappvsvi rw t -144, 445, Ptnoff'* method 357 ptamolytis I
ftyrr's patches plaamopryaL;
and cancer of the uterine PfeiffrrrUa platelet activating fiewr (PAF)
ctrvLr Pfeiffer's bacillus pkoanorphism
pmcccddioides brasilirnsis 02/ Pfeiffer's phenomenon i pdesiomonas
paracoccidnidomyciosiE o il phoooanaphykxiE Ehigelloidcfi '
paracolons 1 phttohypiwmyccBit 619 Pkt medium
|n:it:1 1r1 11.n■ri r_l v.rui 515-16 phage plmropneumonia-like organisms
paralytic polkunydit^ assay 466 CPPI.O)
paramyxemridae , 447 convtraion 465 psiitacosis-lymphogranuloma
parapommis 464 DNA i trachoma (PLT: 422
pararypboid 606 susceptibility test pneumococcus (sir. pneumonia*;
p u m n m i (e« rrulker F iwdn} typing 166 i
paroxysmal cold hemoglobinuria , phagocytosis „ , , I pneumocyEt^ carirui 582,599, 592
disorders of 153 pneumolyMn O 1
parHinysmal nwturaJ bcrooglobinuTi? phagolysosome pneumovirw 447, >
1,14 ' phagosome (vHcuok) ■ pock assay 4.10
p^riiTvindse 447 phenetk system ( fW'linmvdms (infantile paralysis)
parvovirus 431H435, 447, 563 phenols ." *45, ,491
paschen bwiies 421, 431, 1 phenotype mixing, viral 445 abortive
passenger htue , 574 phi-jLlophora 619 eradication of
pasteurelk phleboEomui fitter nonparalytic
antiseptic! Itl phlehotomus :>dj:1 jhii paralytic stage -
boviHptici 331 phlebowrua 447
strainE typr and 491
lepiseptici 331 phosphatase test 607
muLtocida . 331 photochromogens virus 491
ptiLii i.■■i-u yemni:i) phiktotrophi I poliovaocines
pseudetuberculoEis (see yersinia ph^omycetes till polk^iTuSei 491-94
pseudotubeiruloEis) ph^omjtosis, subcutaneous 1 polyacrylamide p i dectmphurcai*
RluI Buftnd test 85,' , 4g4 piournaviridae 447 polykaryucytosis (lyncynuni
pcdiculus humin us corporis (body picomaviruses 432,433, i formation] 1
Idubc) piedra 613 polynvrase chain reaction 591
penidlfinase (beta lactamase) I piedraia hortie 613 polytmuvinis
polyriboajd rih in jl phosphate (P R P )
(WniCiIRwi'' i pigeonpox 46B
jyiiii.ilInn:: 612, antigen
riWs medium *
mamcf&i - pill (see fimbriae) polysaccharide vaedne ■
pentonE pinra 377, 335, Ponders stain
pqilomers 431 Pitman-Moore strain RmtLiie fever 1
peptido^ljnan , pityiiaas vamcciM1(diva wretocim) ul2 pooled human gammaglobulin
peprtKOCD pityiurporum posnLifiil awab postnatal
peprasuepiociMcus n r a b ) 34 2
1 1:■
1 1lIj .rl- (MaJuMna furfur) 612
anaerobius (formerly P putridns) ovale i i posE-transfusioii mcmasrucleoEis
plague powisran virus 523, 531
aiKeharolytku? bubonic ptKviridae . 468
magnus tlomolk- I PPA reaction
preroc. imJd (pestis minor] Ftaunita^Kustner (PK) reaction
tetradiije pneumonic
periodic acid rehiff stain (PAS stain) prophylaxis 11rr-:'i11ir:11i■:ji'■ttaCWn ■ . , 623
611 septi ;(mi r PreiEZr-Mocard bacillus
permeability faemr (PF) plague assay , 44$, 466 p ra tiu n itio n
pernanl swab 342 plaque inhibition test ■ pnwmpove iotifiorn ,'iiunt i
pemndase teEt 107, plaque reduction neutralisation tear primary atypical pneumonia (PAP)
persistent toleraru infection l (FRNT) 522 E3r 397
pertactin___ plaques 466 primary complex 355
pesiii minor plasma cells prion 448, 567

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bSb * te x tb o o k of m ic r o b io lo g y -
traubicula vectotl embryonated eggs 439
tmtsugamushi disease i biological tissue culiurt 439
tuberculin allergy mechanical detective 430
(infecnoo) type hypmen*itivity veiltonellae ■ fixed 556
lubcfeiilnpinTriii. 360 venltairaman-ramakrishnin (VR) general properties
Lull r in dcciciencv medium < gtn*t»es uf ;
tularemia ’ .331 vein cell line ■ 444 growth in cell culture 439
lirriour nH.TT.njs e.L.'t-:r vrmeYfLrioxin (csr vltti itrhxiiii hfmaRgfurirLjicinn of
. verruca vulgaris (human warts) * irtoumpJete 43®
tuifcnntn 46 B verruga peruana i morphology of 431-33
turlock virus 522 vesicular stomatitis virus 454. 522 oncogenic 574-01
TWAR (Taiwan aojte respiratory) vesiculovirus 522, 5l4„ 515 spread in the body
strain 1 v (actor vaccines
i viahic Lncmnl 606 visnj 566
t y t u U l l i m i a n I sll- a t r i k i i M n ) Vi antigan 466 Vogec-Fruskauer (VP) test
ryphoid fever (-see enteric fcser) vibrio
typhoid vaccine alginolyticus volutin granules (Eabes-E-mst
injectable (typhim-Vi) choleraic granules)
Live (lyphoraJ) I classification Von Magnus phenomennn 4,30, 506
ryph UN fever B3, el 1or
endemic (murine m fleabo-me Nap (MM^LTuiable)
w
typhus) « vibrios
epidemic (classical, gaol lever) NCV' (noncholcra) vibrios Vfrgatsuma agar 1
Waldenstrom's matroglobulinamiia 1
recrudescent (Btill-Zinsser differentiation from allied wiiv.iwr i vintf 534
lI]H£Ue) , 41 ? genera Wurth in-hTtdoekky cells I
Tzanck cells WHits, human (verruca vulgaris")
morpholugy
comma wHter, bacteriology of 6G3-6ft5
mimicus i presumptive coliform count 604
l wuitrpn perineum
pajahaettioiynois <
ultrasonic vibration I vcdniFiTH Winerhouir-KfidefLchsen lyndlHtH
uhttviolfl ran 1 vihrion hutyrique i■
undula::[ fever vihrLon sepciquc 1 »eigL-! vaccine I
universal donor weft dM«$e 389, 391
vidarabine 459,
universal Lmmiimsanon nroRminnie Yihcient's angina , 398 V tt-H fa i t w : , 421
(U1P) Vi polysaccharide vaccine, injectable western blot test 592, !
univensnl recipient purified west tule vims 522, 524,526,
ureap(asm* ■ viral diarrhea 635 west:; posnusat swab 342
urc-jplasma urealyticum 39B Virchow’s Lepra (foamy) cells i 'wet ikisriiL^' 597
urease nest < virion 431, 455, 43& whey af^kitinution test 607
useless immunoglobulin vinoids 44$ white graft Tcsponsr
virulence whitlow, herpetic 476
V errhancLUHint of (irDenuurimi]. whooping cough 342, 610
marker antigens (VMA) f : cell strain
vaccine Widal tear' i
boOfltf dime TLduirliun of (rKaltation.)
test 2 wiboti and Blair medium
*sa i
subunit vinia (es)
abnormal replicative eyries 43E Wiskott-Aldrich syndrome
tissue culture wood's lamp
hepatitis h ! artificial 1
assay woolsorter's disease
vaccine piewnlable (tseares (VFD) Wight's stain
vaednia 439. 46B, ( L'uosiny diarrhea 572
varicella (chickpea) -_TK, chemical properties of 432
varicella zoster (V-Z) i classification and nomenclature of N
vtuiuU 468-71, xenografts (formerly Eicreragnft])
variolation Ailtn4tnn of 438 xenopsyila
VtJRL anogtn 379 animal inoculation 439 asna

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Textbook of Pathology
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Mmdalfar and Menon's Oinfcaf Obstetrics [Tenth Edition)
Edfoerf 6y Sarah Capstan & Vkntoa Jaki

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BN S i 250 2808 0
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